Linking multipoint folding and stability with functional regulation in the mitochondrial transmembrane β-barrel Sam50

Domain: biology
Published: 2026-04-02
Editorial outcome: major_revision

Abstract
Mitochondrial outer membrane proteins (β-OMPs) serve as the first line of communication with the
cellular milieu. A crucial β-OMP, the sorting and assembly machinery Sam50, is a 16-stranded
transmembrane β-barrel highly conserved in all eukaryotes. Sam50 dysregulation is lethal; yet,
molecular elements that regulate Sam50 remain poorly understood. Here, we identify and characterize
residues that regulate Sam50 structure and function. Using single-molecule electrophysiology, in vivo
function, and stability measurements, we demonstrate that the POlypeptide-TRansport Associated
domain is dispensable for Sam50. Complete characterization of the folding mechanism using 165
Xaa→Ala substitutions reveals that Sam50 folds through parallel pathways, with at least two transition
states. The folding nucleus is towards the N-terminus, whereas frustrated folding at the C-terminal
region kinetically traps the structure. We correlate this unexpected folding of Sam50 with its SAM-
assisted assembly. Additionally, our per-residue stability measurements show that destabilizing hotspots
in Sam50 are linked to its gating function. Our findings suggest how the dynamic structure of Sam50
offers a functional advantage, with specific residues that regulate folding, stability, and function, also
determine the protein’s sensitivity to mutations.

Introduction
Mitochondria, the site of cellular bioenergetics, are dual-membrane organelles. The
mitochondrial outer membrane (mOM) is uniquely enriched with transmembrane β-barrel OM proteins
(β-OMPs). β-OMPs regulate metabolite flux, transport ions, proteins, and lipids, form mitochondrial-
endoplasmic reticulum (ER) contacts, and maintain mitostasis.1 β-OMPs are nuclear-encoded and
cytosolically synthesized. They are then imported into the intermembrane space (IMS) of mitochondria,
where they undergo a highly regulated directional insertion into the mOM. This β-OMP insertion is
mediated by the Sorting and Assembly Machinery (SAM).2-4 Sam50, the core SAM component, is a 16-
stranded β-OMP that facilitates substrate insertion into the mOM, with one N-terminal POlypeptide
TRansport Associated (POTRA) domain located in the IMS that mediates precursor release (Figure
1a).2,5,6 Yeast Sam50 additionally binds to peripheral subunits Sam35/Sam37 through its cytosolic
loops.3,4 Sam50 differs functionally from ATP-dependent chaperones in its ability to scaffold the folding
of nascent β-OMPs (including itself) independent of electrochemical energy and membrane
composition. In humans, Sam50 also regulates mitochondrial dynamics, and is involved in lipid
scrambling and mitochondrial quality control.1,7,8 These features render Sam50 essential for
mitochondrial biogenesis and mitochondrial integrity, with defects in Sam50 function linked to
mitochondrial disorders.9

Insights into how the complex process of yeast Sam50-mediated β-OMP folding has been
obtained from a stable structural intermediate captured using cryo-EM.3,4 Here, the β1 seam of the
Sam50 transmembrane domain (TMD) associates dynamically with β16 (Figure 1a). This association
allows β1 to template the C→N assembly of nascent β-OMPs into the mOM. The N-terminal lateral
gate (β1-β3) undergoes structural interconversion between gate-closed (resting) and gate-open
(substrate-bound) states, while the POTRA domain and Sam35/37 remain unaltered.3,4,10,11 Interestingly,
however, the thermodynamics regulating such structural dynamics remains poorly understood. More
importantly, the mechanism of how Sam50 itself folds, the folding pathways adopted by Sam50, and the
residue-specific regulation of transition state structures, remain unclear.

Here, we present details linking Sam50 folding, stability, and function. We show that Sam50
TMD folds and functions independent of its POTRA domain, SAM complex, and Sam35/37. The
folding is stepwise, directed by the primary sequence, and proceeds through parallel pathways. The
transition state structures are similar, where strands β3-β6 nucleates folding, followed by β7-β11
assembly. Residues at β13-β15, form non-native interactions, exhibit late assembly, but are vital for
Sam50 function in vivo. Our findings suggest spontaneous membrane insertion of the pre-assembled N-
terminal strands, and a SAM complex-assisted insertion of the C-terminal strands. Folded Sam50 is
destabilized thermodynamically at β4-β6, and this is mechanistically relevant for the “open↔closed”
interconversion of the Sam50 lateral gate during its function. We suggest this stability-function trade-off
as a possible advantage allowing Sam50 to efficiently fold nascent β-barrels, while also performing
additional essential functions in the mitochondrial membrane.

Results
Folding and function of POTRA-less Sam50 resembles full-length Sam50
Sam50 is believed to have evolved from the primordial β-barrel assembly machinery (BamA-
BAM) of Gram-negative bacteria. Here, all extramembranous POTRA domains (except one) are non-
essential for mitochondrial Sam50 function.5 First, we asked if the TMD was sufficient for Sam50
folding, stability, and function. To address this, we generated a POTRA-less variant of yeast Sam50
(Sam50Δ120, only TMD; referred to hereafter as Sam50) (Figure 1a). We examined if the structural and
functional features of the full-length Sam50 (Sam50FL) were retained in POTRA-less Sam50. Similar to
observations with Neurospora crassa Tob55,6 BamA12 and other β-OMPs,13,14 both Sam50FL and
Sam50Δ120 can fold spontaneously in artificial membranes without SAM assistance (Figures S1-S5).
Screens of folding and stability of both proteins in large unilamellar vesicles (LUVs) yielded PC:PE
LUVs (90% DMPC, 10% DMPE) as optimal for path-independent assembly (details in SI Methods,
Table S1).

Folded Sam50 in PC:PE membranes resists proteolysis by proteinase K, and forms a β-rich
structure as revealed by far-UV circular dichroism spectropolarimetry (comparable to NcTob556 and 16-
stranded EcOmpF15). Folded Sam50 also shows a 10 nm blue shift in Trp fluorescence to 336 nm, with
a twofold increase in fluorescence intensity (Figures 1b-e, S1-S6). These observations show that Sam50
can fold into a stable β-barrel independent of its POTRA domain. Sam50 has no identified role in
conducting ions; yet, it can exhibit channel-like behavior in electrophysiology experiments (Figures 1f-
h; S1e-p), with unknown in vivo significance.6,11,16 Comparison of both full-length and POTRA-less
constructs using single molecule electrophysiology6,11, 13,16 revealed channel-like transitions between two
conductance states O1 and O2 (Figure 1f), as reported earlier,11 with the removal of POTRA favoring
the lower conductance O1 state in Sam50Δ120. Both proteins also exhibit similar changes in voltage-
dependent conductance (Figure 1g), and step-wise channel insertion events (Figure 1h). We additionally
validated the in vivo function of Sam50 by complementation of yeast lacking the nuclear-encoded
SAM50. Survival of cells carrying pFL39-SAM50FL and pFL39-SAM50Δ120 was similar in both
fermentable (glucose) and non-fermentable (glycerol) carbon sources at permissive (25 °C, 30 °C) and
non-permissive (37 °C) temperatures (Figure 1i). Taken together, our results show that the TMD is
sufficient for Sam50 folding and function, both in vitro and in vivo.

Sam50 exhibits thermodynamic stability in PC:PE membranes
In β-barrel membrane proteins, several factors determine thermodynamic stability, reversible
folding, and stepwise assembly: (i) the intrinsic ϕ-ψ preferences (β-sheet propensity), (ii) backbone H-
bonding strength of transmembrane residues, and (iii) side chain hydration of lumen-facing residues and
hydropathy of lipid-facing residues.17 The complex interplay between Sam50 and LUVs precludes a
simple interpretation of its thermodynamic parameters. Hence, to deduce the apparent change18,19 in
Gibbs free energy (∆𝐺0) upon Sam50 folding, we monitored Trp fluorescence across 0.67-5.39 M
GdnHCl as the chemical perturbant. We obtained slow microscopic
reversibility and two-state Sam50 (un)folding in lipid:protein ratios of ~1600:1 (~6 proteins per LUV) at
28 °C in PC:PE LUVs (Figure 2a, S7).

Unlike bacterial OMPs,18-22 we found that Sam50 β-barrel is less stable thermodynamically. We
measured a ∆𝐺0,WT of –6.66±0.04 kcal mol-1, a folding cooperativity (M ) of –3.01 kcal mol-1 M-1, and a
mid-point of chemical denaturation (C ) of 2.21±0.01 M (Table S2). Next, we generated a library of
165 Xaa→Ala substitutions in the TMD (Figure 2b), and deduced the contribution of each side chain to
β-barrel stability, by measuring the energetics for each variant (∆𝐺0,Mut) (Figures 2c, S7-S8, Table S2).
The lower entropic cost of introducing Ala (with fewer rotamers) provided us with a per-residue
thermodynamic map of Sam50 (Figures 2d, S9). Here, most substitutions did not alter the overall β-
barrel stability. Despite its moderate stability, we found that the Sam50 structure is tolerant to point
mutations.

Intrinsically destabilizing Sam50 residues (∆∆𝐺0 > +0.45 kcal mol-1) primarily involve Gly, Pro,
and polar residues (Figure 2d, maroon). They are distributed in the β-barrel’s lipid and lumen face,
midplane, and the interface, and are marginally higher at β4-β6. Specifically, Ser, Thr, Asn, and Gln
destabilize Sam50 when they are located at β1-β8 lumen over β9-β16. We inferred that such a polarized
destabilization localized at the N-terminal strands correlates with Sam50 function. Opening of Sam50
lateral gate,4 and repositioning of β1-β3 in the mOM for substrate assembly, requires hinge-like
conformational displacement at β4-β6. Therefore, strategically positioned residues at β4-β6 with
lowered stability (Figure 2d, highlighted region), could facilitate the interconversion of “closed” and
“open” states of Sam50. Supporting our observation, we found only two residues (W240, F455) as
intrinsically stabilizing (∆∆𝐺0 < –0.45 kcal mol-1). Other transmembrane aromatic residues do not alter
Sam50 stability (Table S2), suggesting that the contribution of W240 and F455 is location-specific.
Our experiments show S170 (β4), N175 (β4), N415 (β13), and P462 (β15) are destabilizing hotspots
(Table S2). The folding cooperativity is altered when N175 or P462 are additionally mutated, possibly due
to accumulation of a folding intermediate (Figures S8-S10). These residues form intra-β-barrel
interaction (N415 with L6), or with other SAM complex proteins (P462 with Sam37; S170 with Mdm10),3
and can be functionally relevant. The intrinsic destabilization of N415 affects the stability of the C-
terminal strands during substrate assembly, which is overcome by L6 docking interactions.23 Whether
intrinsic Sam50 destabilization can also promote oligomerization in vivo, remains to be determined.

β-Barrel stability allosterically regulates protein dynamics
Next, we characterize Sam50FL and Sam50Δ120 in PC:PE membranes using all-atom molecular
dynamics (MD) simulations to map the protein’s dynamics (Figures 3, S11-S15). We found larger
spatio-temporal dynamics and conformational states in Sam50FL over Sam50Δ120. This is due to the
intrinsic dynamicity of the POTRA domain, which increases the radius of gyration (R ) (Figures S14-
S15). The root-mean-square fluctuation (RMSF) of the TMD is marginally lower in Sam50FL than
Sam50Δ120 (Figures 3a,c, S13a). Particularly, the strand tilt at β1-β3 (w.r.t. bilayer normal) is lower in
Sam50FL than Sam50Δ120, causing structural compaction at the lateral gate in Sam50FL, while Sam50Δ120
β1-β3 are partly open (Figure 3a). Therefore, POTRA may help allosterically modulate the β1-β3 switch
between gate-open and gate-closed conformations.

WF-Sam50Δ120 (W240/F455→A; intrinsically stabilizing residues) showed a broader free energy
landscape (FEL) than Sam50Δ120, due to larger TMD dynamics (Figure S15). Additionally,
W240/F455→A increases the β-barrel midplane and T3 (P218-P234 linking β6-β7) distance by 0.5-1.5 nm
when compared with Sam50Δ120 (Figures 3b-c, S12-S15). Our simulations revealed that this increase in
dynamicity of T3 of only 18 residues –unlike longer loops (L6: 41 residues; L4: 31 residues)– is due to
A240 (on β7) and A455 (on β15) causing an allosteric effect on T3. Hence, the midplane W240 (β7) and
F455 (β15) anchor the TMD, help form structured extramembranous segments, and are vital for Sam50
structural integrity. The loss in membrane anchoring explains the destabilization we observed when
W240 or F455 is replaced (see Figure 2d).

In contrast, SNNP-Sam50Δ120 (S170/N175/N415/P462→A; intrinsically destabilizing residues) shows
a narrow FEL, with an increase in TMD structural order. Here again, T3 dynamicity is high, suggesting
T3 conformational entropy is sensitive to various thermodynamic perturbations in the TMD (Figures 3b-
c, S12-S15). Our simulations establish that the TMD residues of Sam50 exhibit allosteric modulation of
the dynamics of extramembranous regions, and may thereby influence cytosolic Sam35/37 binding, N-
terminal gating, substrate interaction, or Sam50 function.

Sam50 β-barrel follows parallel pathways for assembly
Next, we studied how per-residue energetics influenced Sam50 folding and structure(s) of the
transition state (TS) ensemble formed during folding. We addressed this by combining ultrafast mixing
with kinetic measurements of the change in Trp fluorescence. Sam50 kinetics is multiphasic. Fits of
change in Trp fluorescence provided three folding (k , k , k ) and two unfolding (k , k ) rate
f1 f2 f3 u1 u2
constants (Figures 4, S16-S18). At [GdnHCl] ≈ C , the (un)folding transitions could not be accurately
m
captured, possibly due to very slow protein rearrangement (see Figures S4-S5, S19).

We observed three kinetic folding and two unfolding rates, which can correspond to sequential
or parallel pathways. Our interrupted (un)folding measurements over different aging times (t ), and
age
[GdnHCl], provide refolding and re-unfolding rates that match the direct (un)folding measurements
(Figures 4a, S20-S21). This supports a parallel assembly mechanism19 for Sam50 in PC:PE membranes.
The re(un)folding amplitudes show no [GdnHCl] dependence, further supporting parallel assembly
pathways. Fits of the WT chevron profiles to two-state models provided a total free energy change for
the kinetic process ∆G 0,WT that was larger than ∆G0,WT (Figure 4a, Tables S3-S4). The measured
kin,sum C F
differences in ∆G0,WT and ∆G 0,WT arise from structural and bond rearrangements in the TS, and the
F kin,sum
multiple (un)folding rates (Figures 4a,d). These bond rearrangement kinetics also explain why Sam50
(un)folding equilibrates slowly.

The parallel pathway for Sam50 β-barrel formation involves the fast (forms TS ) and slow
1
(forms TS ) pathways, with accumulation of an early intermediate I (corresponding to k ) during
2 E f2
folding (Figures 4a,d, S21). For residues above the β-barrel midplane (cytosolic side), rates obtained for
I matched the fast pathway, whereas those for residues below the midplane (IMS side) matched the
E
slow pathway (Figures S22-S23). From this correlation, we propose that I may resemble an on-
E
pathway early folding intermediate that accumulates prior to TS formation, and links both the pathways
(Figure 4d). It is also likely that structural rearrangements during Sam50 folding are uncoupled from
major energy changes, progressing instead via slow strand and loop rearrangements. The Tanford β
value (β )  0.5 (Table S4), and supports a half-open partially compact β-barrel during folding.
T

Structures of the transition states formed during Sam50 assembly
For simple two-state transitions, the structure of the TS can be deduced from an experimental
estimation called -value analysis.24,25 This measures the effect each side chain substitution has on the
(un)folding energetics. In Sam50, the unfolding kinetics showed a stronger [GdnHCl] dependence
(Figures 4a-b, S22-S24). Additionally, the two-state profiles attain rapid equilibrium at high [GdnHCl]
(see Figure S5). Hence, we measured -values for the fast ( ) and slow ( ) assembly pathways
F1 F2
from the unfolding rate constants, by interpolation (k , k ) (Figures 4b-c, S24). Next, using
u1-5.5M u2-4.5M
these rate constants, we deduced the change in unfolding free energy (∆∆𝐺0 ) at each side chain (Table
‡−N
S3). Finally, by comparing ∆∆𝐺0 with ∆Δ𝐺0, we obtained the per-residue  map (Figures 5a-b, S25-
‡−N F F
S26). To increase confidence in the  values and minimize estimation errors, we only considered
F
variants with ∆∆𝐺0 > ±0.05 kcal mol-1.
F

-values define the local structure of each residue in the transition state, with  ranging from
F
~0 (unfolded in TS) to ~1 (folded in TS). Values outside the 0-1 range ( = –0.3 to –3.01;  =
NN NN
+1.301 to +4.0) represent non-native interactions formed transiently during folding, but are absent in the
end-state structure. To obtain a simplified map of how Sam50 folds, we depicted the  on a two-
F1/2
dimensional topology diagram (Figures 5a-b, S25-S26). We colored each residue that was structured in
the TS as blue, unstructured as red, and forming non-native interactions as grey. The results showed
strands β3-β6 as structured in the TS in both pathways, suggesting that β3-β6 assembly is a common
early nucleation state during Sam50 folding (Figure 5, zone (i)). Residues forming native-like
interactions are also seen in β7-β11 (Figure 5, zone (ii)), interspersed with non-native interactions,
suggesting that these strands are also partially structured during Sam50 assembly. Non-native
interactions localized at β14-β16 (Figure 5, zone (iii)) revealed the assembly of C-terminal strands as
the rate-limiting step in Sam50 folding.

In the fast pathway ( ; TS1), several residues of β6-β12 were unfolded or formed non-native
F1
contacts (Figure 5a, zone (ii)). Such non-native contacts can form when transfer of charged residues of
L4/L6 across the membrane is energetically disfavored. In contrast, in the slow pathway ( ; TS2), β7-
F2
L4-β8 and β10-L6-β12 assembled correctly (Figure 5b, zone (ii)). Here, several residues are “folded” at
β9-β10, facilitating assembly of this hairpin, which can then lower the energy barrier for membrane
transfer of L4/L6. In both pathways, folding is nucleated at β3-β6. Subsequently, the fast and slow
pathways are demarcated by the extent of local structure formed at the mid-barrel (β7-L4-β8 and β10-
L6-β12).

The β of ~0.5 can stem from structural compaction following non-native interactions at the C-
terminal β-strands. In vivo, the SAM complex is believed to template structuring of C-terminal strands,
and β-barrel closure.4 In our experiments, these processes progress slowly in the absence of SAM,
also explaining the transient hysteresis during early time points of our thermodynamic measurements (see
Figures S4-S5). Whether SAM complex also accelerates the membrane transfer of L4/L6 is, however,
unclear.

Put together, we find that assembly at β3-β6 is spontaneous, and may be nucleated in the IMS in
the holdase-bound state or the membrane vicinity (see Figures S31-S32). The post-folding structural
destabilization we measured at β4-β6 (Figure 2d; highlighted) reveals a kinetic role for these β-strands
in nucleating Sam50 folding. Folding is retained in Sam50Δ120, emphasizing the intrinsic ability of the β-
barrel and extramembranous POTRA domains to fold independently.

Trade-off between residues vital for Sam50 folding and function
Our thermodynamics and kinetics measurements identified key residues that cause frustrated
folding, impede rapid β-barrel formation, and affect Sam50 scaffold stability (Figures 2d, 5a-b, S9, S25-
S26)). To assess if these residues were retained evolutionarily for superior function, we examined their
in vivo relevance. We monitored survival of yeast YPH499 sam50::ade2,26 complemented with pFL39-
SAM50FL, pFL39-SAM50Δ120, and their mutants, under non-permissive conditions (37 °C, non-
fermentable carbon source, oxidative stress). We found Sam50 (FL and Δ120) as tolerant to point
mutations (Figure S27). Therefore, we next determined the functional relevance of TMD strands, T3,
and L4/L6, through multiple Xaa→Ala substitutions.

Surprisingly, complementation was successful only for a subset of variants (Figure S27). Non-
permissive mutants mapped to residues at the midplane and cytosolic interface of β10-β14
(L315(β10)/Y390(β12)/T440(β14); K341/V420) (Figure S28). Our in vitro measurements showed that β10
(L315) contributes to mid-β-barrel assembly, and β10-β13 is essential for membrane transfer of L6.
Therefore, we propose that these multiple mutations negatively affect the correct positioning of L6 and
subsequent β15-L6-L7-Sam35 interactions. L315 (β10) is essential in both Sam50FL and Sam50Δ120,
supporting a POTRA-independent functional role for this residue. Others, such as K341 and V420, located
at the neck of L6 and L7, can control loop dynamics and regulate pore size of the Sam50 lumen. T440
can stabilize β15-Sam35 interaction3 at the bilayer interface through vicinal residues (e.g., E437). The
other Sam50 subset that did not support yeast survival included I185(β5)/I277(β8)/G443(β14)→A. We
attributed this to a combined loss in structural features involving T3 (I185) and L4 (I277) dynamics, and
β14 curvature nucleated at G443 (the central hinge residue with unusual ϕ- values; discussed later).
These mutations were lethal also in Sam50FL, and POTRA could not rescue this lethality.

Taken together, residues displaying frustration in Sam50 folding also appear vital for its
biogenesis and its function in the cell. Many of these residues are conserved between BamAEc, Sam50Sc,
and human Sam50 (Figures S29-S30). Our study revealed that, despite the successful evolutionary
divergence of Sam50 for adaptation to the mitochondrial membrane and its interactome in the specific
eukaryotic cell, the retention of select residues from bacteria to humans emphasizes their functional
importance. For example, β14-GT443 is necessary for the hinge-like structural gating motion of Sam50.2,4
Despite causing folding errors, sequences such as β14-β16, or the stereo-selective positioning of
residues such as G443, are retained evolutionarily for their vital role in Sam50 function in vivo.

Methods
Cloning, mutant library generation, and protein production
Saccharomyces cerevisiae SAM50 cloned in pGEM4 was a kind gift from Prof. Doron Rapaport
(University of Tübingen, Germany). The complete gene was PCR amplified and re-cloned in pET3a
between NdeI and BamHI, downstream of the T7 promoter. Full-length pET3a–SAM50 was used as the
template for generating the POTRA deleted variant (pET3a–SAM50Δ120). This was achieved through
conventional mutagenesis and gene manipulation, wherein the first 360 nucleotides at the 5′ end of
SAM50 were excised, and the gene re-ligated in-frame to obtain the POTRA–less variant. Both pET3a–
SAM50FL and pET3a–SAM50Δ120 were used as the templates for generating all the mutant variants
described in this study.

The modeled structure of Sam5010 was used as the guide to map residues constituting the
transmembrane domain of this protein. The codon for each non-Ala transmembrane residue of
SAM50Δ120was mutated systematically to Ala using transfer-PCR.S19,43 A total of 165 variants of
Sam50Δ120 were thus generated, and incorporation of the mutation was verified in each case using
Sanger sequencing. Chemically competent Escherichia coli C41 cells were transformed independently
with pET3a–SAM50Δ120 and its 165 mutant variants, and protein expression in the form of inclusion
bodies was achieved upon induction for 4 h with 1 mM (final) isopropyl β-D-thiogalactopyranoside.

Inclusion bodies were extracted and purified (>85% purity) by treatment with 2.75% Triton X-100
followed by 0.5% sodium deoxycholate.14,19,44 The preparations for Sam50Δ120 was additionally
subjected to fast protein liquid chromatography (FPLC) denaturing purification on a 20 mL HiPrep SP-
FF cation ion exchange column using an increasing 1 M NaCl gradient (Buffer B) prepared in 50 mM
acetate buffer pH 5.0 containing 8 M urea (Buffer A). Fractions containing the purest form of Sam50Δ120
were pooled, and dialyzed against water to remove urea, residual buffers, and salts. All protein
preparations (from inclusion bodies and chromatographic purification) were stored at –86 °C as
lyophilized powders. All 166 variants were produced tag-free and without a mitochondrial targeting
sequence(s), and checked for protein integrity on 12% SDS-PAGE before use.

Preparation of large unilamellar vesicles
Lipid vesicles contained the 14-C saturated phosphatidylcholine DMPC (1,2-dimyristoyl-sn-
glycero-3-phosphocholine) doped with a defined concentration (10%) of the 14-C non-lamellar lipid
DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine). For folding screens, the 16-C
phosphatidylcholine DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and cholesterol were also
used. Defined molar volumes of DMPC, DMPE, DPPC, and cholesterol, in required ratios and
concentrations, were aliquoted in glass vials. Chloroform, methanol, and trace solvents in the lipid
suspensions were removed by drying under a stream of dry N gas. These lipid films were dried further
2
by lyophilization for 4 h, following which they were stored at –86 °C and used within one week of
preparation.

Lipid resuspension (final concentration = 40 mM) was achieved by hydrating the dried film in
the desired buffer (listed in Table S1), followed by alternating heating–vortexing cycles at 54 °C for 45
min to obtain a suspension of 40 mM lipid. Large unilamellar vesicles (LUVs) of uniform 100 nm
diameter were prepared using 40-50 cycles of extrusion at 35 °C through a 0.1 μm polycarbonate
membrane using the mini-extruder assembly (Avanti Polar Lipids, Inc.).

Preparation of Sam50Δ120 stocks
Different conditions were screened to obtain the buffer, lipid composition, lipid:protein ratios,
temperature, pH, and dilution factor that supported complete folding of Sam50 (details in SI Methods).
125 μM Sam50Δ120 was prepared in 8.0 M GdnHCl in 50 mM citrate buffer pH 5.0 and 10 mM DTT
(unfolded stock). Protein quantification was achieved using a molar extinction coefficient for Sam50Δ120
of 53400 M-1 cm-1 at 280 nm (https://www.expasy.org/resources/protparam). In the first step, this
unfolded protein stock was diluted 10-fold to achieve 12.5 μM Sam50Δ120 in 20 mM LUVs (90 %
DMPC + 10 % DMPE), 50 mM citrate buffer pH 5.0, 4.0 M GdnHCl, and LPR = 1600:1. Next, this
reaction was diluted 5-fold either in buffer (to prepare the folded stock) or 4.0 M GdnHCl (to prepare
the unfolded stock). Both stock preparations were incubated for 12-14 h at 28 °C, with rotational
mixing. These folded and unfolded stocks contained final concentrations of 2.5 µM protein, 4 mM
LUVs (90 % DMPC + 10 % DMPE), 50 mM citrate buffer pH 5.0, 0.2 mM DTT, and 0.8 M GdnHCl
(folded stock) or 4.0 M GdnHCl (unfolded stock). The final LPR was 1600:1. These preparations were
diluted an additional 6-fold in the equilibrium thermodynamics and kinetics experiments. This stepwise
protocol was also followed for the preparation of Sam50FL.

Pulse proteolysis
Unlike bacterial outer membrane proteins, Sam50Δ120 does not exhibit differential mobility on
cold SDS-PAGE. Therefore, to assess folding efficiency, the folded protein stock prepared as described
above, was subjected to proteolysis using the robust protease proteinase K (PK).45 Protein stocks were
diluted with 50 mM citrate buffer pH 5.0 and PK was diluted in PK buffer. PK was added so as to
achieve different protein:PK ratio (5:1 and 1:3.5) in the final reaction. Samples were drawn at different
time intervals and the reaction quenched by addition of phenylmethylsulfonyl fluoride (final
concentration = 8 mM or 1.2 mM). Samples were loaded on 12% SDS-PAGE and checked for protease
protection, by comparing with the appropriate controls (Figures 1c, S1b).

Far-UV circular dichroism (CD) measurements
The secondary structure content of the folded protein was assessed on a JASCO J-815 far-UV
CD spectropolarimeter (JASCO Inc., Japan). The folded stock protein (2.5 μM protein folded in 4 mM
LUVs) was subjected to wavelength scans from 210–260 nm at 1 nm increments and scan speed of 50
nm/min, at 28 °C, using a quartz cuvette of 1 mm path length. The contribution from buffer, GdnHCl
and lipid were nullified using appropriate blank corrections, and the raw ellipticity values were
smoothened using the means–movement method.45 The averaged raw ellipticity (θ, mdeg) from three
accumulations was converted to molar ellipticity (ME) at each wavelength,19 and plotted (Figure 1d).
Per-residue ellipticity was calculated by dividing the ME at each wavelength by the total number of
residues. The signal at ~255-260 nm was set to 0 mdeg, to nullify effects of scattering from LUV
preparations and plotted as normalized θ (mdeg) for the mutants (Figure S6).

Transmission electron microscopy
To check the integrity and size of vesicles, TEM images were obtained. Protein-free vesicles
(0.5 mg/ml) recovered after rotational mixing (similar to the stock preparations) at 1100 rpm for 14 h at
28 °C, were immobilized by coating on a carbon–coated copper grid, followed by staining with 2%
uranyl acetate, washing with milliQ, and drying at 28 °C for 4 h. TEM images were obtained on a
ThermoFisher Scientific TalosTM F200S (S)TEM operated at accelerated voltage of 200 kV at –196 °C.

Electrophysiology measurements
Sam50 is a β-barrel with a central luminal pore with a sufficiently large diameter to facilitate the
movement of ions. We used this feature of the protein to check if Sam50Δ120 can form a complete
functional β-barrel without its soluble N-terminal POTRA domain. To study the channel forming
properties, both Sam50FL and Sam50Δ120 were first assembled in LDAO micelles by rapid 10–fold
dilution of the unfolded protein in 6.0 M GdnHCl, and 10.0 mM DTT, to achieve 25 μM protein, 65
mM LDAO, 10 mM DTT, 100 mM NaCl, in 50 mM phosphate buffer pH 7.2. Samples were incubated
for 12 h at 4 °C using rotational mixing. Post-incubation, samples were centrifuged at 16,600 x g for 1 h
to remove trace amounts of aggregates. The supernatant was quantified, mixed with 0.1% cholesterol
and 1% triton X-100, and incubated on ice for 15-20 min, before use in the experiment. 0.5-1.5 μL of
this sample was added on the cis side of a single DiPhPC bilayer (containing 0.1% cholesterol) painted
across a 150 μm aperture of a polysulfone cup.44 Both sides of the bilayer membrane contained 10 mM
HEPES pH 7.4, 5 mM CaCl , and 1 M KCl. The potential across the black lipid membrane was
2
maintained at +10 mV using an 8-pole Bessel filter (Warner Instruments, Harvard Apparatus Inc.), and
Ag/AgCl electrodes. Protein was added only after a tight membrane with a near-zero conductance was
obtained.

Insertion of the protein in the DiPhPC membrane was achieved through slow stirring. For
capturing the stepwise insertion events, the protein was added to the cis side of the assembly, and the
solution was magnetically stirred, allowing for various protein molecules to be inserted into the DiPhPC
membrane consecutively. A holding potential of +10 mV was maintained throughout. For voltage ramp
measurements (both single channel and multichannel experiments), the applied voltage across the
membrane was varied systematically from 0 mV → +100 mV → –100 mV → 0 mV (at 3.33 mV/s), and
the voltage-dependent characteristics of the Sam50 β-barrels were acquired using a filtering frequency
of 400 Hz. Similarly, for holding voltage experiments, the potential across the bilayer membrane was
maintained at a defined voltage, and the system was monitored for stable channel-forming behavior. The
voltage was increased stepwise by 10 mV, to capture channel closing events. Channel-like flickering
behavior of both proteins at holding voltages of +10 mV and +50 mV were captured. Recordings for
multichannel voltage-ramps were carried out with a 15 kHz filtering frequency. The data was acquired
using Clampex 10.3.1.5 (Molecular Devices, LLC) and processed using Clampfit 10.6.2.2 (Molecular
Devices, LLC).

Equilibrium folding and unfolding
Equilibrium thermodynamic parameters were deduced from the fluorescence emission profiles
of the seven intrinsic tryptophans of Sam50Δ120. Progress of the reaction was followed over time using a
microplate reader (MicroMax accessory of FluoroMax-4, Horiba Jobin Yvon Inc., or the SpectraMax
M5 from Molecular Devices LLC). Folded and unfolded protein stocks were titrated into a defined 60-
point gradient of GdnHCl by 6-fold dilution (0.42 μM protein, 0.67 mM PC:PE lipid, in 50 mM citrate
buffer pH 5.0). The final [GdnHCl] varied from 0.67–5.39. Samples were prepared in a 96 well
microtiter plate, with a final reaction volume was 200 μl in each well. The LPR of 1600:1 was
maintained throughout. The reaction was incubated at 28 °C, and the progress was monitored for up to
48 h, using a λ = 280 nm, and emission spectra recorded in the top read mode from 310–400 nm, at 1
ex
nm increments. Equilibrium was achieved in 24 h, and the spectra showed no further change with time.
All spectra were corrected for buffer, GdnHCl, and lipid contributions, and the average wavelength
(<λ>)32 was calculated from the corrected 24- and 48-h spectra using Equation 1.

< 𝜆 > = ∑𝜆𝑖𝐼𝑖 / ∑𝐼𝑖
Equation 1

Here, λ is the ith wavelength, and I is the corresponding fluorescence intensity at λ.
i i i

Sam50Δ120 and all its 165 Ala variants show a two-state transition between the folded and
unfolded states, with no detectable intermediates. The <λ> values plotted against the respective GdnHCl
concentrations was therefore fitted to a two-state equation (Equation 2) assuming a linear dependence of
the transition between folded and unfolded states, on the GdnHCl concentration.24,32

< 𝜆 > = 𝑦 = (𝑚U [𝐷]+𝑦U)+ (𝑚F [𝐷]+ 𝑦F) 𝑒𝑥𝑝(− ∆𝐺F 0+𝑀F[𝐷] / 𝑅𝑇) / 1+ 𝑒𝑥𝑝(− ∆𝐺F 0+𝑀F[𝐷] / 𝑅𝑇)
Equation 2

Here, 𝑦 and 𝑦 are the intercepts and m and m are the slopes of the pre- and post-transition baselines,
F U F U
respectively. The denaturant (GdnHCl) concentration is D, and M is a measure of the folding
F
cooperativity. We obtained a M = –3.01 kcal mol-1 M-1 for Sam50Δ120 and its mutants, from global
F
analysis of all the data. Here, Δ𝐺0 is the apparent change in Gibbs free energy of folding, R is the
TF
universal gas constant (1.987 x 10-3 kcal mol-1 K-1), and T is the experiment temperature in kelvin. 𝑄 is
the normalization factor for the emission intensity, and is given as (Equation 3):

𝑄 = ∑𝐼i,F / ∑𝐼i,U
Equation 3

where 𝐼 and 𝐼 are the fluorescence intensities of the fully folded and unfolded states of the protein,
i,F i,U
respectively. We used 𝑄 = 1 for our analysis. The midpoint of chemical denaturation 𝐶 = ∆𝐺0 / 𝑀 .
m F F
The ∆𝐺0,WT (for Sam50Δ120) and ∆𝐺0,MUT (for each Sam50Δ120 Ala variant) was used to derive the change in
F F
free energy due to the mutation (ΔΔ𝐺0) (Equation 4). Two experimental replicates of 60-point titrations
F
were carried out, as they offer robustness for deriving thermodynamic parameters.

∆∆𝐺0 = ∆𝐺0,WT −∆𝐺0,MUT
F F F
Equation 4

All-atom molecular dynamics simulation
AlphaFold–predicted model of Sam50FL and Sam50Δ120 modeled using the SWISS-MODEL web
tool was used as starting structures. The mutant variants WF–Sam50Δ120 (W240/F455→A, destabilized
Sam50Δ120 β-barrel), SNNP–Sam50Δ120 (S170/N175/N415/P462→A, stabilized Sam50Δ120 β-barrel),
Sam50Δ120–W240A, Sam50Δ120–F455A, and Sam50Δ120–P462A, were created with CHARMM-GUI.
The input system for GROMACS was generated with CHARMM36m force field, and all-atom MD
simulations carried out using GROMACS v2020.5, v2023.2 and v2024.2, as reported earlier.19 Briefly,
the structure was assembled in a rectangular box of lipid bilayer membrane containing 90% DMPC +
10% DMPE. 30 Å and 20 Å water layers were maintained on both sides of the bilayer with Sam50FL and
Sam50Δ120 respectively for hydration. Per leaflet, ~100 lipids were used (90 DMPC and 10 DMPE). NpT
(isothermal–isobaric) ensemble with constant particle number, pressure, and temperature (28 °C), were
used during the simulation. The complete system parameters are tabulated below for Sam50FL and
Sam50Δ120. The output file with 6 steps of energy minimization (restraints were reduced at each step44),
was used for a 500-550 ns production run with no restraints. A 500 ns simulation is sufficient to capture
the protein dynamics being investigated and enhanced sampling is not needed in this case. A step size of
2 fs was used. At least three rounds of simulations were carried out with independent protein–lipid
assemblies to ensure that the initial configuration does not influence the simulation results (confirmed
from analyses presented in Figures S11, S14). The first 0.1-1.0 ns data usually presented a glitch in
equilibration (illustrated in Figure S11 inset); therefore, data from the first 10 ns was excluded from the
analysis. The root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), and free
energy landscape (FEL) for the protein were calculated .19 Additionally, distances were calculated
between P228 Cα of turn 3 (T3) and the β-barrel midplane (W240 Cα) to determine the dynamicity of T3.

System parameters:
Parameter Sam50FL Sam50Δ120 Description
Box Type Rectangle Rectangle
Crystal Type TETTRAGONAL TETRAGONAL
System Size A 90.647903 91.120789 Dimension along the A (X) axis
B 90.647903 91.120789 Dimension along the B (Y) axis
C 195.68452 105.98163 Dimension along the C (Z) axis
Crystal Angle Alpha 90 90 Angle between the axis B and C
Beta 90 90 Angle between the axis A and C
Gamma 90 90 Angle between the axis A and B
# of Lipids on Top 100 101
on Bottom 100 100
Z Centre -25.238618 2.9691994 Centre of the system along the Z axis

Material and scripts pertaining to the MD simulations are provided as a separate supplementary file
(compressed).

Real time millisecond (un)folding kinetics
Millisecond folding and unfolding kinetics were measured on a SX20 stopped-flow spectrometer
(Applied Photophysics Ltd., UK) equipped with asymmetric mixing, dual detection, and a 20 μl flow
cell. Folding kinetics of Sam50Δ120 were captured by 6-fold dilution of the unfolded protein stock 2.5
µM protein, 4 mM LUVs (90 % DMPC + 10 % DMPE), 4.0 M GdnHCl (unfolded stock) with 50 mM
citrate buffer pH 5.0 and varying concentrations of GdnHCl to achieve final GdnHCl concentrations
varying from 0.67–1.80 M. Unfolding was initiated by 6-fold dilution of the folded protein stock with
defined concentrations of GdnHCl from 3.0–6.8 M, to achieve final concentrations of 0.42 μM protein,
0.67 mM PC:PE lipid, in 50 mM citrate buffer pH 5.0, and varying [GdnHCl]. All experiments were
carried out at 28 °C. Both folding and unfolding kinetics were followed by change in Trp fluorescence
using a λ = 280 nm, and total emission was recorded on a PMT detector equipped with a 360 nm
ex
bandpass filter. Ten technical repeats were collected for each [GdnHCl], and averaged using Pro-Data
Viewer (Applied Photophysics Ltd., UK). Data for the folding kinetics was fitted to a triple exponential
rise function (Equation 5a) to obtain the folding rate constants (k , k , k ) and corresponding
f1 f2 f3
amplitudes (A , A , A ). Data for the unfolding kinetics were fitted to a double exponential decay
f1 f2 f3
function (Equation 5b) to derive the unfolding rate constants (k , k ) and corresponding amplitudes
u1 u2
(A , A ).
u1 u2

y = A *exp(–k *t) + A *exp(–k *t) + A *exp(–k *t) + c
f1 f1 f2 f2 f3 f3
Equation 5a

y = A *exp(–k *t) + A *exp(–k *t) + c
u1 u1 u2 u2
Equation 5b

Here, t is the time, and c is the fitting constant. The initial 50 ms data carried artifacts due to convection
effects from mixing of viscous solutions, and was therefore excluded from the fits. Plots of the natural
logarithm of the rates at each [GdnHCl] provided the chevron plot, which was fitted to Equation 6, and
the change in kinetic free energy ∆𝐺0 for Sam50Δ120 and a few representative Ala variants were
kin
calculated using Equation 7.

ln𝑘 = ln(exp(ln𝑘 +𝑚 [𝐷])+exp(ln𝑘 +𝑚 [𝐷]))
obs F F U U
Equation 6

∆𝐺0 = −𝑅𝑇(ln 𝑘0 −ln 𝑘0)
kin F U
Equation 7

Here, [D] represents the denaturant concentration, ln𝑘 is the observed rate constant, ln𝑘 and ln𝑘
𝑜𝑏𝑠 F U
are folding and unfolding rate constants in the absence of denaturant, respectively, and m and m are
F U
the slopes for folding and unfolding arms, respectively. R is the universal gas constant (1.987 x 10-3 kcal
mol-1 K-1), and T is the experiment temperature in kelvin.

Unfolding kinetics were carried out for all 165 Ala variants of Sam50Δ120. Data for at least 15
GdnHCl concentrations were recorded to derive reliable kinetic parameters of unfolding. Each chevron
unfolding limb generated by plotting the natural logarithm of k and k against [GdnHCl] for each
u1 u2
Sam50Δ120 Ala variant was fitted to a two-state model using Equation 8.

ln𝑘 = 𝑚 ∗[𝐷] + ln𝑘
U U U
Equation 8

A shared m = 1.02 kcal mol-1 M-1 and m = 1.30 kcal mol-1 M-1 was used for nearly all the variants
U1 U2
(see Table S3), and rate constants (𝑘5.5M and 𝑘4.5M) were obtained at 5.5 M GdnHCl (for k ) and 4.5 M
U1 U2 U1
GdnHCl (for k ) by interpolation,46 to minimize estimation errors. These GdnHCl concentrations were
U2
chosen, as they were the approximate midpoints for each arm. The Sam50Δ120 variants deviating from
this shared m were grouped as low and high m , and analyzed separately (data compiled in Table S3).
U U

Interrupted (un)folding measurements
For interrupted folding experiments, the unfolded protein stock comprising 5.0 or 8.75 µM
protein, 8.0 or 14.0 mM LUVs (90% DMPC + 10% DMPE), 50 mM citrate buffer pH 5.0, and 6.0 M or
4.0 M GdnHCl, respectively, was incubated overnight at 28 °C with rotational mixing. In the first step
of the interrupted folding measurements, the unfolded stock was diluted 6-fold or 3.5-fold with buffer to
initiate folding of Sam50Δ120, and the reaction was allowed to age for defined delay times (t ). Folding
age
was interrupted by 2-fold or 6-fold dilution of this reaction in unfolding buffer (3.75/ 4.25/ 4.5/ 5.0/ 5.5
M GdnHCl in 50 mM citrate buffer pH 5.0), while simultaneously capturing the unfolding kinetics
using change in Trp fluorescence (as described above). Three technical replicates were recorded at 28
°C at each t and each final [GdnHCl]. Each decay profile was processed independently by fitting to
age
double exponential decay function (Equation 5b), to derive k and k for each t .
u1 u2 age

For interrupted unfolding experiments, the folded protein stock comprising 8.75 µM protein, 14
mM LUVs (90% DMPC + 10% DMPE), 50 mM citrate buffer pH 5.0, and 0.8 M GdnHCl, was
incubated overnight at 28 °C with rotational mixing at 1100 rpm. In the first step of the interrupted
unfolding measurements, the folded protein stock was diluted 3.5-fold with 4.0 M GdnHCl (final
concentration in the delay loop) to initiate unfolding. The reaction was aged for defined delay times
(t ). Unfolding was interrupted by a 6-fold dilution of this reaction with buffer (final GdnHCl in the
age
cuvette was 0.67 M). The refolding kinetics were captured using change in Trp fluorescence (as
described above), and three technical replicates were recorded at 28 °C for each t . Each decay profile
age
was processed independently by fitting to a triple exponential rise function (Equation 5a), to derive k
f1
,
k and k for each t .
f2 f3 age

Φ–value analysis
The effect of each mutation in the Sam50Δ120 sequence was used to deduce the structure of the
transition state (‡) formed during protein (un)folding, using Φ-value analysis. First, the change in free
energy of unfolding from the folded (N) to the transition state (‡) was calculated directly for the mutant
with respect to WT (∆∆𝐺0 ) for each chevron unfolding limb using Equation 9.
‡−N

∆∆𝐺0 = −RT ln( 𝑘 U x,WT / 𝑘x,MUT)
‡−N U
Equation 9

Here, 𝑘x (𝑘5.5M or 𝑘4.5M for each unfolding limb) was obtained by interpolation of the data at x = 5.5 M
U U1 U2
(for k ) and 4.5 M (for k ) GdnHCl, to minimize errors arising from extrapolation to 0 M GdnHCl.25
U1 U2
Φ–value analysis, to deduce the transition state structure, was then calculated from both the equilibrium
and kinetic data using Equation 10. Here, Φ was obtained for each unfolding limb.
U

Φ = ∆∆𝐺0 ‡−N / −∆∆𝐺0
U F
Equation 10

Finally, Φ was derived from Φ using the correlation Φ = 1 – Φ .
F U F U

In vivo yeast survival assays
Saccharomyces cerevisiae strain YPH499 with the chromosomal copy of SAM50 deleted
(YPH499 sam50::ade2 [Yep352-MET25pr-Sam50-CYC1t]; MATa ura3-52 lys2-801_amber ade2-
101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1) complemented by the YEpSAM50 plasmid expressing Sam50
constitutively (control),26 was a kind gift from Dr. Nils Wiedemann, University of Freiburg, Germany.
The shuffle strain (also obtained from Dr. Wiedemann) was created by the Wiedemann laboratory in
collaboration with Dr. Bernard Guiard (Centre de Génétique Moléculaire, CNRS, France).26 pFL39 E.
coli–yeast shuttle vector carrying the SAM50 gene11 was also provided by Dr. Nils Wiedemann. Here,
the yeast SAM50 gene is expressed constitutively and in low copy numbers by being cloned between its
native promotor and native terminator sequences.11 pFL39–SAM50Δ120 was generated by overlapping
PCR.43 Additionally, point mutants were generated both for SAM50FL and SAM50Δ120 by transfer-PCR43
or cloned from pET3a–SAM50 to pFL39 using assembly PCR. All genetic manipulations were
confirmed in pET3a and pFL39 by Sanger sequencing.

The pFL39-SAM50 full length, Δ120, and all mutant variants were transformed in the YPH499
sam50::ade2 strain,26 followed by plasmid shuffling on 5-fluoroorotic acid (5-FOA) media.10 Cells that
successfully underwent plasmid shuffling were identified by their selective growth only in yeast
synthetic dropout media w/o Trp (these cells failed to grow in dropout media w/o Trp and Ura).

Serial spot dilution assays to measure survival of YPH499 sam50::ade2 (SAM50 deletion strain)
carrying pFL39-SAM50 were carried out using drop test.44 Briefly, cells were grown in the synthetic
dropout medium w/o Trp, until an OD = 1.5 ± 0.1 was achieved. Cells were harvested, washed twice
600
with milliQ, and resuspended to an OD = 0.5. Cells were then serially diluted 10–fold (fold-dilution =
600
0, 10, 100, 1000, 10000) (Figure 1i) and 25–fold (Figure S27), and spotted on (i) YPD (1% (w/v) yeast
extract, 2% (w/v) bactopeptone, 2% (w/v) glucose), (ii) YPG (1% (w/v) yeast extract, 2% (w/v)
bactopeptone, 3% (w/v) glycerol), and (iii) YPDA (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 2%
(w/v) glucose, 40 mM acetic acid) plates additionally containing 2% agar, and incubated at 25 °C, 30
°C, and 37 °C. Plates were imaged at 48 h (YPD), 55/72 h (YPG), 72 h (YPDA). Here, YPH499
sam50::ade2 strain transformed with yeast episomal plasmid YEpSAM50FL (expressing SAM50FL
constitutively) was used as the control. Complementation of Control (YPH499 sam50::ade2 with
YEpSAM50FL) was then done with pFL39-SAM50FL (expressing full-length SAM50 constitutively),
followed by curing of YEpSAM50FL. Similarly, complementation of Control (YPH499 sam50::ade2 with
YEpSAM50FL) was done with pFL39-SAM50Δ120 (expressing POTRA-less SAM50 constitutively),
followed by curing of YEpSAM50FL.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "regulation of the  OMPs is an important and long-standing question. Ravi and his/her colleagues investigated the\nthermodynamic and kinetics of an N-terminus truncated Sam50 (Sam50 1-120) variants in the lipid bilayer environment to\nexhibit their thermodynamic stability, path-independent folding, and stepwise β-barrel assembly process. The study offers\ninsights into how Sam50’s folding process is regulated by its β-strands and loops, although interesting, no external factors,\nsuch as the SAM complex, were considered in this analysis. More comprehensive investigations, such as those including\nthe SAM complex, would be necessary to gain a more complete understanding of Sam50’s folding dynamics. While the\nauthors have made significant strides in mapping key residues involved in Sam50 folding, these results may not fully\naddress the broader  OMP-folding-regulation mechanism. Additionally, the manuscript’s writing expression could be\nimproved to enhance clarity and ensure broader accessibility to the findings. A thorough revision of the manuscript is\nrecommended to improve readability. Below are some detailed suggestions for improvement:\nMajor:\n1. Previous findings support that the assembly of the  OMP needs the help of the SAM/BAM machine. Ravi et al. found that\nthe Sam50 can spontaneously insert the N-terminal strands into the membrane, even without the association of the SAM\ncomplex. The author should provide more interpretations for this very unexpected finding.\n2. Why must the authors use the Sam50 1-120 rather than a full-length Sam50? I would like to know if they can get similar\nresults when they use FL-Sam50 as a target, as well as the Tom40 or Mdm10.\n3. Page 4 line 16, a fully folded  OMP may be exhibited by a crosslinking strategy (Qiu, Jian et al. Cell. 2013 Aug\n1;154(3):596-608. doi: 10.1016/j.cell.2013.06.033.). Considering there is no SAM complex in the LUV, the author should\nprovide evidence showing that this spontaneous insertion system really works.\n4. Mammalian Sam50 is related to the neurological disease in mammals rather than the fungal Sam50. Also, the Mdm10 is\njust a potential linker in the fungal; there are currently no homologs of Mdm10 in human cells. So, the authors should\ncarefully present the sentence in the abstract: “Our findings thus show how conformational buffering of Sam50 structure-\nfunction handles evolved for superior mito-biogenesis and mito-ER interactome and pave the way for designing knowledge-\nbased peptide therapeutics for neurological ailments.”\n5. The model is confusing. 1) The evidence for proving the beta-strand self-folding when binding the small Tim chaperone\nneeds to be more characterized. 2) The Step 3 presented here is involved in the hybrid of the nascent beta-strand and the\ntemplate beta-strand. This step may represent a key process for understanding the beta-OPM folding, but details are absent\nin the zoom-in figures. 3) The thermodynamics and kinetics measurements in the paper did not contain SAM complex.\nAlthough the authors have included an in vivo relevance for some chosen Sam50 variants, they’d better only provide this\nmodel figure just to show the spontaneous folding process of Sam50.\n6. The POTRA domain is a conserved part in the 16-beta-strands OMPs, such as BamA in bacteria and OPE80 in\nchloroplast. The function of the POTRA domain is currently elusive. Could the authors figure out some interpretation for the\nfunction of the POTRA domain from the data and analysis in Figure 3? Also, could the authors give some speculations as to\nwhy different 16-beta-strands OMPs chose different numbers of POTRA domain?\nMinor:\n1. Page 4, lines 15, 31, 32, and 36, It is not clear about the difference between the “path-independent assembly” and the\n“path-dependent assembly”. The authors should add some definitions of the two types of assembly.\n2. Fig 1g, what’s the difference between the control and the FL? It seems like the same construct but with a different\nphenotype in some situations.\n3. Fig 1f, the authors should include a negative control such as a liposome without protein.\n4. Figure 1c. The author did not label the band, which would confuse them. Additionally, the accumulation of the ~27 kDa\nspecies cannot be observed clearly (lane 5). Furthermore, the folded Sam50 should not be digested by PK without PMSF\n(lane 6).\n5. Figure 3b. This MDS should be further confirmed by a single molecular experiment, for instance, smFRET.\n6. Page 9 line 11, are the conserved residues in BamAEc or other beta-OMPs also involved in their folding regulation?\nThe authors should add discussion here.\n7. Figure 3a, the results relevant to the all-atom MDS of Sam50 120 and full-length Sam50 are not clear. The author should\nfigure out which structure represents the Sam50 120, as well as the full-length Sam50.\n8. In Figures 2 and 4, the author should label the number of the beta-strand for a better understanding of readers.\n9. Page 22 line 19, the “red diamond” may be the “red circles”.\n10. Page 6, lines 36-39, it’s difficult for readers to understand how the data in Figure 4a support the description: “a parallel\nmechanism for Sam50Δ120 assembly in PC: PE membranes”. More detailed interpretations of the data are suggested here.\n11. Page 7, lines 3-20. It is also difficult to understand the relationship among the “multiple pathways of folding”,\n“parallel pathway”, and the “TS1 and TS2”. The authors should provide more accurate interpretations for this part.", "reviewer_2": "Referee report for Ravi et al: In this paper, the authors present a series of biophysical and biochemical assays and\nsimulations characterizing the folding and stability of the Sam50 protein in model membranes. They further perform in vivo\nassays in yeast to assess lethal sets of residue mutations. The introduction establishes that this mitochondrial membrane\nprotein is essential for cells and notes that defects in its function contribute to mitochondrial disorders. Overall, the authors\nhave collected large datasets to comprehensively assess single-point residue mutations (none of which are lethal), some\n‘multipoint’ mutations, as well as assays that identify folding transition states. While these datasets will be useful to\nresearchers studying the protein Sam50, the paper is extremely technical and does not establish a clear or coherent story\nabout how all of this data is broadly useful or insightful to non-experts or a broad audience in biology or biophysics, or how it\nwill be useful for therapeutics or disease. A persistent issue is the use of words that are not standard in biophysics or are\nmisused, along with statements that do not make sense. There are many aspects of the system that are not clear even after\nreading the paper several times. For example, is the folding ever studied in the membrane in the presence of the SAM\nchaperone? Is it the Sam50 dimer (Sam50b and Sam50a)? Or just one subunit? A major issue is that the collected results\nare not integrated together. The MD simulations in particular are almost completely divorced from the study of folding of the\nprotein, as they only compare energy landscapes of structures under mutations, showing rather minor effects. Many of the\nfigures do not support strong conclusions (Fig 3 and 4), and I am not sure if Fig 5 is speculation or data.\nGeneral issues:\nThe abstract is already very difficult to parse. What does ‘regulators’ mean? MDS is not a standard acronym, even MD would\nbe better. 165 variants of Sam50 is actually 165 mutations to the Sam50b subunit? Multipoint is not a familiar or standard\nword in biophysics: at multiple residues in the sequence. I am not sure why the word mutation is rarely used. ‘This\nmetastable structure’? Which metastable structure is this referring to? And again, what are ‘microscopic regulators’? What is\nconformational buffering? What are structure-function handles?\nIn short, the authors do not seem to be entirely clear what they are trying to say, and have leaned on broad generalizations\nthat muddy what approach they have taken and what they have learned. The word regulators needs to be replaced with\nsomething specific, like residues, secondary structures, or something that reflects what they tested and learned.\nThese sentences contain more information: “We show that Sam50 folds through multipoint nucleation and N-terminal\nstructuring, whereas C-terminal frustrated folding forms kinetically-trapped intermediates. This unexpected folding correlates\nmechanistically with SAM-assisted in vivo assembly”. However they are still confusing. When does the C-terminal folding\noccur, do they see this in the membrane? The fact that this is even undergoing folding in the membrane is not clear, nor is it\nclear that they look at only one subunit, or that the SAM machinery is known to assist in protein folding within membranes.\nThe authors describe the structure as “metastable” throughout the text. This has a strict thermodynamic meaning: this\nstructure is not the free energy minimum, but can persist for some time range because it has a barrier separating it from the\nfree energy minimum. This is therefore not the most favorable equilibrium structure. When do they show this? It appears to\nbe asserted without support. They seem to be abusing the word metastable, or at least haven’t actually provided evidence in\nthe text of the metastability. To the contrary, that the protein folds to a functional state on its own despite many mutations\nseems to indicate overall stability.\nThe MD simulation results are largely divorced from the rest of the study. Despite having extensive residue-level\nexperimental data, the simulations are not directly compared to the experiments in any way. The main conclusion is that the\nfree energy landscape becomes wider as a function of the RMSD, but they already showed that that protein has a wider\nRMSD because it has an extra domain, so it will automatically sample more values. These are not insightful plots about the\nfunction. They have a proposed folding mechanism and it is not interrogated using the atomistic simulations.\nThe authors refer to a ‘kinetic free energy’ (P7L4) and ‘energetic contributions from both kinetic and thermodynamic\ncomponents’ (P10L31). This is misleading. Free energies are by definition equilibrium measurements that have no kinetic\ncomponent. If they extracted them from experiments that also measured kinetics, that is fine, but don’t assign this confusing\nlanguage to a free energy.\nThe following specific parts in the manuscript require further clarification and/or restructuring:\nOn page 3 it is mentioned that the SAM complex helps to fold Sam50. However, all of the results seem to be Sam50 folding\nby itself in the membrane, rather than folding with the assistance of the SAM chaperone activity.\nFigure 4: I am not sure how to interpret these plots, they seem more like supplementary information. There are a series of\npoints and lines, and what I am supposed to expect vs what they see is not clear from the plot. Comparison of (d) and (e)\nrequires staring at these sequences and picking out variations, none of which are highlighted and made clear. Yes there are\ndifferences between them, what do I learn from this?\nFigure 5: Are these results or speculation? Where did the Tims come from? These proteins were never mentioned and were\nnot included in the experiments.\nPage 3: what do the superscripts mean on the names of proteins? Some are Sam50, one is SAM, why?\nP4 L38: Protein folding is bimolecular? What does this mean? Bimolecular reactions typically refer to two species at well-\nmixed concentrations binding to one another through space. Folding is a polymer dynamics problem. At no point in the SI\ndoes it show a bimolecular treatment of the observed dynamics. Sums of exponentials are not indicative of a bimolecular\nreaction (which has units of M-1s-1 for rates).\nP5 L5: Surface area in units of kcal /mol /M, this is completely unfamiliar to non-experts\nFig 3a overlaid structures are very difficult to understand. What are we supposed to be seeing here?\nFig 3d: what do we learn from these plots? It is unsurprising that FE landscape is broader with respect to RMSD when\nthere’s a large domain added that increases the RMSD.\nFig 3d D120 landscape: are those two bumps supposed to be the metastable and stable states?\nFig 3c: Are these curves statistically significantly different? Error shading?\nStatements that are unclear and are not supported by data:\n'Beta barrel stability allosterically regulates protein dynamics'? How does ‘stability’ allosterically regulate dynamics? I have\nno idea what this is trying to say.\nP6 L22: “Our simulations establish how intrinsically destabilizing residues modulate signal propagation across the\nmembrane, with zonal changes (cytosolic Sam35/37 binding, N-terminal gating, substrate interaction) allosterically\npropagating through the β-barrel, to alter IMS dynamics and regulate Sam50 function”. What are zonal changes? Again the\nword regulate is used but it is nonspecific, regulate how? How am I to interpret ‘modulate signal propagation’? What signal?\nWhy do the authors fit to a triple exponential for folding and a double exponential for unfolding? Where did this choice for the\nnumber of rates come from?\nS19: Why do the kinetics and equilibrium measurements overlay so well (“excellent overlay”) when at the same time,\n“Sam50Δ120 undergoes slow rearrangement (possibly through multiple pathways of near-equal populations), which cannot\nbe captured in the rapid kinetics measurements, but is evident in the equilibrium titrations.”?", "reviewer_3": "This manuscript by Ravi et al. presents a detailed and multifaceted investigation into the folding mechanism, stability, and\nfunctional regulation of the mitochondrial outer membrane protein Sam50. By integrating thermodynamic profiling,\nultrafast folding kinetics, molecular dynamics simulations, and in vivo functional assays, the authors provide detailed insights into the\nfolding landscape and structure-function relationships of Sam50.\nThis study addresses a significant gap in our understanding of mitochondrial β-barrel assembly. Major Strengths include the\ncomprehensive approach combining experimental and computational tools. Also, the mapping of thermodynamically\n(de)stabilizing hotspots and their in vivo validation enhances the translational relevance of the work. I have a few points that\nneed to be addressed before considering publication.\nThe folding studies were performed in a PC:PE (90:10) lipid system. While this is experimentally tractable, it doesn't fully\nmimic the native mitochondrial outer membrane environment. The authors mention the use of \"native-like\" conditions, but\nthis lipid mixture is distinct from actual mitochondrial membranes. The manuscript would benefit from a discussion on how\nthe choice of lipid composition may influence Sam50 folding energetics and the generalizability of the findings to in vivo\nconditions.\nThe manuscript notes the potential for structure-based peptidotherapeutic development. However, this important point is not\ndeveloped in detail. The authors should elaborate on which structural or energetic features of Sam50 could be rationally\ntargeted for therapeutic design and how their findings might inform future efforts. As it stands, the therapeutic implications are\nspeculative without further context.\nThe figure captions are too long and include large amounts of methodological and interpretive information, which would be\nbetter placed in the main text. The paper would benefit from concise captions and improved integration of the relevant details\ninto the Results and Discussion sections.\nMinor points:\nPage 5: I didn't understand the units of surface area (\"3.01 kcal/mol*M\"). Can the authors clarify?\nPage 5: \"other transmembrane Phe(18), Tyr(6), and Trp(3), do not stabilize the...\" - maybe say residues after Phe, Tyr, and\nTrp?\nPage 12: \"evisage\" - should be envision\nPage 12: \"drug designing\" - should be design", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "Ravi et al have addressed a part of my concerns. This study offers insights into how Sam50’s folding process is regulated by its β-strands and loops. The additional revisions have strengthened the manuscript; however, despite the authors' efforts to address the reviewers' comments, some aspects still require further clarification. Given that it is the first time showing that Sam50 can fold successfully in membranes in an in vitro system, without assistance from the SAM complex, the authors should provide more compelling evidence showing that this spontaneous insertion system works (see below). Moreover, they should better interpret whether this spontaneous insertion process could occur in the mitochondrial outer membrane under physiological conditions.\nMajor:\n1. Although the authors showed four results that tried to prove the Sam501-120 can be spontaneously inserted into the\nartificial membrane, there are also things that need to be validated.\na)Figure 1c. The folded/unfolded Sam50Δ1–120 cannot be digested by proteinase K in the presence of PMSF, since PMSF should fully inhibit PK. As the 27-kDa band appears under similar conditions in Figure S1b, the authors should show this digestion result in Figure 1c without PMSF to clarify this point.\nb)Figure 1d. I suggest separating the two datasets in Figure 1d into two individual panels to enhance readability.\nc)Figure 1f. From the perspective of experimental completeness, a protein-free planar bilayer membrane should be included as a negative control in the electrophysiological recordings, even if no current is expected. This would help confirm that any observed current is indeed attributable to Sam50.\nd)The electrophysiological experiment is intended to demonstrate that the refolded Sam50 is functional. However, ScSam50 has not been reported to act as an ion channel. Therefore, additional evidence is needed to confirm that Sam50 was fully folded in the authors’ system. Alternative functional assays or structural confirmation would be helpful.\n2. The folding behavior of β-OMPs in artificial membranes may not reflect their folding in mitochondrial outer membranes.\nGiven that Sam50 is essential for yeast viability (Paschen et al., Nature, 2003; Kozjak et al., J Biol Chem, 2003; Gentle et al.,\nJ Cell Biol, 2004), the authors should discuss whether β-OMPs can spontaneously fold in mitochondria in the absence of the\nSAM complex. Furthermore, the discussion should be expanded to include other β-OMPs mentioned in the rebuttal letter,\nsuch as Tom40 and VDACs.\n3. In the rebuttal letter, the authors mention three cryo-EM studies that captured the SAM complex “in action” — with fully\ncaptured Mdm10, fully folded Tom40, and partially folded Tom40. However, in Figure 6 (panel “2”), only the interaction\nresidues of “SNNP” within the SAM and SAM–Mdm10 structures are mapped. The authors should also address: 1) The\n“SNNP” motif in the two SAM–Tom40 structures. 2) The corresponding residues in the AlphaFold-predicted\nSAM–Porin complex.\nMinor:\n1. Figure 6, the author should use “a, b, c” as icons rather than “1, 2, 3”.\n2. Throughout the manuscript, replace “mOMP” with the more widely accepted term “β-OMP.\n3. The term “insertase” is used to describe Sam50 in the revised version. However, this term is typically reserved for\nYidC/Alb3/Oxa1 family proteins, which mediate the insertion and folding of α-helical membrane proteins. The authors should\nreconsider this terminology or provide justification for its use in this context.\n4. The role of the POTRA domain should be discussed more thoroughly in the Discussion section.\n5. The English language in the manuscript remains awkward in places and sometimes difficult to follow. Careful\nproofreading and editing are needed to improve the clarity, flow, and overall readability of the text.", "reviewer_2": "The authors have significantly improved the text through rewriting their manuscript, with more precise language, improved integration of the distinct parts into a coherent narrative, and improved figure clarity. Additionally, they have clarified many of the details and confusing language. However, we still have issues that we feel should be addressed:\n1. Regarding the electrophysiology experiments:\na. While it’s clear that the properly folded Sam50 forms a membrane channel, it is not established how a channel current and conductance switching would be indicative of “full functionality” as claimed in the electrophysiology results.\nb. The characterization in the text of the data in fig 1f seems off: the text says both FL and d120 have reduction from ~2-3 to ~1 nS at >+-50mV. Figure 1f shows a change from above 3 to below 3 nS for d120 rather inconsistently with applied voltage, appearing briefly for descending voltage >~200mV, and again for increasing negative voltage from around -400mV to nearly 0. The FL is similar, but with conductance uniformly lower by ~1nS. Additionally, the d120 variant appears to have a third conductance state not seen in the FL trace.\nc. Overall, these data seem not to contribute much to the rest of the (very detailed) study of the folding and structure of\nSam50.\n2. Regarding the molecular dynamics simulations:\na. The first paragraph of the section “β-Barrel stability allosterically regulates protein dynamics” concludes that “POTRA can regulate gate dynamics, and may drive the β1-β3 switch…” This might match the more classical definition of allostery, but it’s difficult to understand how β-barrel stability regulating gate dynamics would be considered allostery.\nb. Our prior comment on error shading in fig 3c seems to have been misinterpreted; it should be possible to show the error\non the mean for the RMSF curves in 3c, e.g. by bootstrapping, so that one can see to what extent the different portions of the\ncurves are different within statistical error.\nc. Caption of 3b says that FL to d120 results in a decrease in T3 “dynamicity” by >1nm. I would think dynamicity would relate to changes or fluctuations. Neither of those two traces show much dynamicity, the change from FL to d120 just shows a change in a rather static distance.\nd. Fig 3c: The emphasis on turn 3 seems to show that while FL and d120 do not fluctuate much here, the two mutants do,\nwhich agrees with the result of 3b. Loop 4 seems to be shown as a counter example where no change occurs in mutants.\nThis could be made more clear in the figure/legend.\ne. Fig 3e is still not informative. How do these FEL connect to the point about conformational gate opening/closing. They\nreport ‘a broader FEL’ in WF-d120. Despite a FEL directly reporting on the relative probability of sampled configurations,\nthey are not used in this way. The color bars also do not seem to agree with the labels on the z-axis, where d120 reaches\n9kJ/mol by eye, but is colored orange. If the FEL is not a main point, as the authors acknowledged in their response, then\nwhy are they being shown in the main text?\n3. In the author's response, they state “Yes, by and large, free energies are equilibrium measurements. However, for\nmembrane proteins, it has been demonstrated through several studies that kinetic components also contribute to the\nmeasured free energy”. This suggests a misunderstanding of what free energy is. We are not arguing that kinetic trapping\ncannot occur in protein folding, nor that this can impact how measurements report on the probability of sampled states.\nHowever, do not refer to it as a free energy when that is defined only for an equilibrium ensemble. If kinetic (time-dependent)\nelements impact the measured distribution of sampled states, then one is not measuring a free energy. This is non-negotiable. When the authors abuse precise, thermodynamic terminology, it undermines the study. Free energies are by definition controlled entirely by the Boltzmann distribution, and not by any time-dependent factors. The reworded statement does now avoid making this assertion. However, one could argue that there is no need to use the word ‘kinetic stability’,\nwhen kinetic or time-dependent on its own will suffice, although this is not an essential change. If hysteresis occurs, this\ncannot be an equilibrium transition between states, as equilibrium transitions are not dependent on paths/history. Or at least\nthe measurements are not capable of sampling the equilibrium transitions.\na. In the SI, it is remarked that the kinetic free energy is “deduced” from fits to SI equation 6, which does not contain that\nvariable on its own. Is it defined from the ratio kU/kF being taken equal to an effective folding dissociation constant?\n4. We appreciate that the authors removed the word metastable. Despite this modification the authors insist in their response\nthat “we disagree with the Reviewer’s comment that a metastable state is unfavorable”. It is by its very definition not the free\nenergy minimum, which means that it is indeed unfavorable relative to the thermodynamic free energy minimum. This is\ntrue whether it is metastable in state space (i.e. a metastable liquid relative to a solid) or metastable within a free energy\nlandscape of an equilibrium ensemble, where a higher energy minimum is metastable because of barriers separating it from\nneighboring states. This minimum on the FEL can only be termed metastable if it is not the global free energy minimum.\n5. Fig 4b and text say that unfolding rates were obtained from interpolation, which seems to be taking the value of the linear\nfit at a particular concentration of GdnHCl (5.5M and 4.5M). Why those specific concentrations?\n6. Fig S31 is a helpful illustration of the kinetic model of folding in LUVs that plays a central role in the paper; the authors\nmight consider having this as part of figure 4, where the folding and unfolding rate measurements are shown. Also regarding\nFig S31: There are rates for folding to the transition states, and unfolding from the final structure to the transition states, but\nnot for folding from transition states to final state. Where are the rates that connect the transition state to the folded state?\nHow do they know that the unfolding from the folded state returns them to the transition state and not to the previous\nintermediate or the disordered/adsorbed state?\n7. Fig 6 caption says G443→A is not tolerated and causes loss in cell viability, but wasn’t it established earlier that no single\npoint substitutions resulted in loss of cell viability?\n8. In the referee response, they state “The proposed folding mechanism derived from kinetics measurements is distinct from\nthe dynamics we obtain for the post-folded structure in our MD simulations, and there is nothing to interrogate in this.” How is\nthere nothing to interrogate when they seem to be stating that the simulations are in disagreement with the experimental\nmeasurements?", "reviewer_3": "I am satisfied with the authors' response to the issues I raised.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Manuscript reference number: NCOMMS-25-06253-T\nRevised Title: Linking multipoint folding and stability with functional regulation in the\nmitochondrial outer membrane protein insertase.\nWe thank the editor and the Reviewers for their time and for the opportunity to resubmit our\nwork. We present our extensively revised manuscript in line with the Reviewers’ suggestions.\nWe take this opportunity to emphasize the importance of our findings. Several studies\nexist on how soluble proteins fold, which, combined with their structures, have together led to de\nnovo design of these molecules. Studies on membrane protein folding, particularly the β-barrels,\nhowever, are extremely limited. Here again, information is available only for the bacterial\nproteins. Thus far, no study has attempted to map the precise processes involved in the\nstepwise folding of any eukaryotic β-barrel. Ours is the first to report it.\nWe acknowledge that our work involves artificial membranes and non-native conditions.\nYet, studies have consistently demonstrated how invaluable in vitro findings remain for\nmembrane proteins, and we present three such recent examples involving OMPs:\n1. Protein-lipid charge interactions control the folding of outer membrane proteins into\nasymmetric membranes. Nat Chem 2023 15(12):1754-1764.\n2. The translocation assembly module (TAM) catalyzes the assembly of bacterial outer\nmembrane proteins in vitro. Nat Commun 2024 15(1):7246.\n3. Conformational plasticity of mitochondrial VDAC2 controls the kinetics of its interaction with\ncytosolic proteins. Sci Adv 2025 11(17):eadv4410.\nAn in vitro biophysical characterization is done with a specific purpose: here, we draw\nparallels between the mechanisms we capture for Sam50 folding and stability, to that\nhypothesized to occur in the cell. Our study maps the folding events of Sam50 using the widely\naccepted Φ-value analysis. The advantage of our approach is providing structures of the\nintermediates that are not possible to capture in vivo, despite the currently available\ntechnologies. We have additionally correlated folding events with the stability of this protein and\nlinked it to cell survival. With this, we obtained new insights into why Sam50 exhibits specific\nbiochemical traits. Three such major findings are:\n1. Sam50 structurally interconverts between “gate-closed” and “gate-open” states. We now\nshow how this transition occurs, and provide a molecular and energetic basis for it.\n2. There are only three cryo-EM studies thus far which could capture the SAM complex “in\naction” – two with fully folded Mdm10 (Takeda et a., Nature 2021), and Tom40 (Wang et al.,\nScience 2021), and one with a partially folded Tom40 (Takeda et al., Nat. Struct. Mol. Biol.\n2023). No study has captured how Sam50 itself folds. Ours is the first.\n3. We prove –for the very first time– that Sam50 can fold successfully in membranes, without\nassistance from SAM complex. We also identify how Sam50 possesses regions that are a\nbottleneck for self-assembly: the C-terminal strands that form non-native interactions.\nAll scientific approaches have their limitations and demand deviation from true in vivo –\nbe it fluorophore labeling, cryoEM samples, modified cell lines, mouse models for human\nsystems– immediately rendering them non-native. Given how no one has attempted to study the\nfolding of this very essential mitochondrial protein, we hope that you would appreciate the\nmagnitude of our efforts, the breakthrough we have achieved in mapping the stepwise assembly\nof Sam50, identifying stabilizing elements, and linking both Sam50 thermodynamics and kinetics\nwith its function in vivo, and accept our study in Nature Communications.\nResponse page 1\nReviewers' comments and Author responses:\nIn this response file, the Reviewers’ comments are in blue, and our responses, in black.\nWherever substantial changes were made in line with the Reviewer’s comments, we present the\nrevised text as-is for your ready reference, along with our response.\nReviewer #1 (Remarks to the Author):\nRegulation of the bOMPs is an important and long-standing question. Ravi and his/her\ncolleagues investigated the thermodynamic and kinetics of an N-terminus truncated Sam50\n(Sam50D1-120) variants in the lipid bilayer environment to exhibit their thermodynamic stability,\npath-independent folding, and stepwise β-barrel assembly process. The study offers insights\ninto how Sam50’s folding process is regulated by its β-strands and loops, although interesting,\nno external factors, such as the SAM complex, were considered in this analysis. More\ncomprehensive investigations, such as those including the SAM complex, would be necessary\nto gain a more complete understanding of Sam50’s folding dynamics.\nAuthor response: We thank the reviewer for appreciating our work and for sharing the\nsuggestions. We specifically chose our study on Sam50 without its auxiliary factors, so that we\ncan successfully map the step-wise folding events of this protein, and identify residues that are\nvital for its stability and function. Our aim here is to obtain precise insights into the regulatory\nelements, which would be extremely difficult to accurately determine if this protein were present\nas a complex. We finally propose how our findings link to in vivo events. To clarify that we are\nstudying only Sam50, we have now added this sentence in the Introduction (page 3, lines 29-\n31):\nWe show that Sam50 TMD folds and functions independent of its POTRA\ndomain, SAM complex, and Sam35/37.\nWe are certain that our findings will pave the way for a more comprehensive understanding of\nSAM complex assembly in vivo.\nWhile the authors have made significant strides in mapping key residues involved in Sam50\nfolding, these results may not fully address the broader bOMP-folding-regulation mechanism.\nAuthor response: We understand the Reviewer’s skepticism. However, studies on β-barrel\nmOMPs are extremely limited, and only three structures are presently available that provide\nsnapshots of how these proteins assemble. Furthermore, while end-state structures can still be\npredicted for β-barrel mOMPs through AI tools, how this structure is achieved is still unsolved for\nmembrane proteins in general, and mOMPs in particular. To this end, our current study aims to\nbridge this immense void in our understanding of mOMP folding-function-regulation mechanism,\nby obtaining detailed information at the residue-level for the mitochondrial Sam50 β-barrel. We\nuse tools and approaches well-established for soluble proteins, to understand stepwise events\nin Sam50 folding, stability of the folded structure, and how both energetic contributions link to\nthe function of this protein. We are confident that our study will inspire broader research on\nmany other vital OMPs.\nAdditionally, the manuscript’s writing expression could be improved to enhance clarity and\nensure broader accessibility to the findings. A thorough revision of the manuscript is\nrecommended to improve readability.\nAuthor response: We welcome the suggestion, and apologize that the earlier submission was\nmore technical. We have now revised the manuscript extensively to retain a clear storyline with\nbroader accessibility and clarity.\nResponse page 2\nBelow are some detailed suggestions for improvement:\nMajor:\n1. Previous findings support that the assembly of the OMP needs the help of the SAM/BAM\nmachine. Ravi et al. found that the Sam50 can spontaneously insert the N-terminal strands into\nthe membrane, even without the association of the SAM complex. The author should provide\nmore interpretations for this very unexpected finding.\nAuthor response: We welcome the suggestion. In fact, a similar assumption was made for\nbacterial β-barrels, that they could only be assembled correctly by the BAM complex. SAM-\nindependent unassisted folding of other mitochondrial OMPs (including yeast Por1, mouse\nVDAC1, and the three human VDACs 1/2/3) have been demonstrated in artificial detergents,\nbicelles, and vesicles. Hence, our finding that Sam50 can fold without the help of SAM/BAM is\nnot unexpected. As suggested by the Reviewer, we now include a statement to this effect on\npage 4, lines 9-11, with the relevant citations:\nAs seen with BamA11 and other mOMPs,12,13 Sam50 (FL and Δ120) can fold\nspontaneously in artificial membranes without SAM assistance.\n2. Why must the authors use the Sam501-120 rather than a full-length Sam50? I would like to\nknow if they can get similar results when they use FL-Sam50 as a target, as well as the Tom40\nor Mdm10.\nAuthor response: Earlier studies indicate that the POTRA domain is redundant, and non-\nessential for Sam50 function (Cell 2008 132(6):1011-24). Given how we intended to understand\nthe folding and stability of Sam50 without any auxiliary factors, we reasoned that POTRA would\nalso be an auxiliary factor. Removing the soluble POTRA domain will decrease any interference\nin our measurements from the extramembranous region, thereby helping us to precisely\ninterpret the role of transmembrane residues in the folding of Sam50. We have succinctly\njustified our approach on page 4, lines 5-7:\nWe asked if the TMD was sufficient for Sam50 folding, stability, and function. To\naddress this, we generated a POTRA-less variant of yeast Sam50 […]\nOther studies from our lab on Sam50FL, human Tom40, human VDAC2, and bacterial BamA\nreveal multiple folding pathways for each protein. Some show unique assembly mechanisms,\nwhile others are similar to the Sam50Δ120 pathways we report here. These findings are currently\nin different stages of being published (under review, under revision). We have not worked on\nMdm10 (since it is present only in mitochondria of lower eukaryotes, and absent in human\nmitochondria), but we hypothesize that spontaneous membrane insertion and path-independent\nfolding are features that this mOMP also possesses.\n3. Page 4 line 16, a fully folded OMP may be exhibited by a crosslinking strategy (Qiu, Jian et\nal. Cell. 2013 Aug 1;154(3):596-608. doi: 10.1016/j.cell.2013.06.033.). Considering there is no\nSAM complex in the LUV, the author should provide evidence showing that this spontaneous\ninsertion system really works.\nAuthor response: We agree that cross-linking is one strategy to confirm folding. While this is a\ngood approach in vivo, this does not necessarily guarantee that other parts of the β-barrel are\ndefinitely folded in vitro. In our experiments, to confirm that Sam50 indeed folds spontaneously,\nwe used four different experimental approaches that could be tested simultaneously on Sam50-\nFL, Sam50-Δ120, or any of the Ala variants:\n1. Resistance to proteolysis by a robust non-specific protease such as proteinase K\n(Figure 1c). Here, an unfolded/ membrane-adsorbed/ partially folded protein is rapidly digested\nby proteinase K (shown in the same gel).\nResponse page 3\n2. β-Rich secondary structure content confirmed using far-UV circular dichroism (CD)\nspectropolarimetry (Figure 1d). The structural content is comparable to reported\nellipticity values for another 16-stranded bacterial β-barrel.\n3. Change in Trp fluorescence intensity (2-fold increase) and emission maxima (10-nm\nblue-shifted emission) (Figure 1d, inset).\n4. Single-channel electrophysiology of these artificially folded samples shows gating\ncharacteristics that match previous reports which studied reconstituted SAM complex\n(Figure 1f).\nPut together, these results (described on page 4, lines 14-28, and Figure 1) confirm that the\nSam50 proteins we are characterizing do indeed fold and achieve a functionally competent\nstate.\n4. Mammalian Sam50 is related to the neurological disease in mammals rather than the fungal\nSam50. Also, the Mdm10 is just a potential linker in the fungal; there are currently no homologs\nof Mdm10 in human cells. So, the authors should carefully present the sentence in the abstract:\n“Our findings thus show how conformational buffering of Sam50 structure-\nfunction handles evolved for superior mito-biogenesis and mito-ER interactome and pave the way for designing knowledge-\nbased peptide therapeutics for neurological ailments.”\nAuthor response: We agree with the Reviewer. Hence, we have reworded the abstract and the\nconclusion sections as follows:\nAbstract (page 2, lines 14-17):\nOur findings suggest how the dynamic structure of Sam50 offers a functional\nadvantage in vivo, with selective residues that affect the folding, stability, and\nfunction, correlating directly with the protein’s sensitivity to mutations.\nConclusions (page 11, lines 34-38):\nThe Sam50 destabilization we detect upon mutation of key residues can\nsubstantially alter SAM complex function, affecting mitochondrial health. The\nidentification of residues tailored for stability and functional efficacy for vital\nproteins, such as what we report for Sam50, can unveil mechanistic correlations\nof mOMPs under both health and disease states, furthering the design of possible\ntherapeutic molecules.\n5. The model is confusing.\nAuthor response: We have now simplified the model we present. We have divided the figure\ninto three segments in Figure 6:\nScheme 1 provides what we have mapped from our -value analysis.\nScheme 2 illustrates residues we identify as essential for Sam50, and how it\nrelates to the important interactions that these residues establish (derived from\npreviously reported structures).\nScheme 3 focuses specifically on the functional importance of the intrinsically\ndestabilized β4-β6 we identify, on the conformational interconversion Sam50\nundergoes. The figure also illustrates the importance of β14-G443.\n1) The evidence for proving the beta-strand self-folding when binding the small Tim chaperone\nneeds to be more characterized.\nAuthor response: We have now edited this figure to only reflect our observations from our\nexperiments. The illustration involving small Tim chaperons has now been removed.\n2) The Step 3 presented here is involved in the hybrid of the nascent beta-strand and the\ntemplate beta-strand. This step may represent a key process for understanding the beta-OPM\nfolding, but details are absent in the zoom-in figures.\nResponse page 4\nAuthor response: Since this information was not obtained directly from our experiments, we\nhave now removed it from the main figure. However, we agree with the Reviewer that this hybrid\nbarrel formation is a key step in mOMP insertion in vivo. Hence, we have moved this figure to\nthe supplementary information (now Figure S32), and included a zoomed-in image of Step 3.\n3) The thermodynamics and kinetics measurements in the paper did not contain SAM complex.\nAlthough the authors have included an in vivo relevance for some chosen Sam50 variants,\nthey’d better only provide this model figure just to show the spontaneous folding process of\nSam50.\nAuthor response: We agree. We have removed all reference to the SAM complex in this figure,\nand have now shown only the unassisted assembly mechanism of Sam50 that we map from our\nstudy.\n6. The POTRA domain is a conserved part in the 16-beta-strands OMPs, such as BamA in\nbacteria and OPE80 in chloroplast. The function of the POTRA domain is currently elusive.\nCould the authors figure out some interpretation for the function of the POTRA domain from the\ndata and analysis in Figure 3? Also, could the authors give some speculations as to\nwhy different 16-beta-strands OMPs chose different numbers of POTRA domain?\nAuthor response: The question on Figure 3 is a very interesting one! Our in vitro\nmeasurements (Figure 1c-f) suggest that POTRA is likely a redundant feature of yeast Sam50.\nWe see no measurable difference in yeast survival under permissive and non-permissive growth\nconditions, despite POTRA deletion (Figure 1g). Hence, this change appears well-tolerated by\nthe cell. Our conclusion is supported by a previous study (Cell 2008 132(6):1011-24). However,\nfrom our MD simulations (Figure 3), we find that the presence of POTRA structurally compacts\nthe transmembrane domain by altering the strand tilt angle, particularly at β1-β3. We believe\nthat this may alter the efficiency with which the transmembrane domain transitions between\n“gate-closed” and “gate-open” states, thereby influencing the binding and/or release of a\nprecursor protein in mitochondria. A previous study also suggests that POTRA may play a role\nin precursor release (Mol Biol Cell 2011 22(16):2823-33). Putting together our observations,\nwe\nhave now proposed the following in our revised manuscript (page 5, lines 37-40):\nParticularly, the strand tilt at β1-β3 (w.r.t. bilayer normal) is lower in Sam50FL\nthan Sam50Δ120, causing structural compaction at the lateral gate in Sam50FL,\nwhile Sam50Δ120 β1-β3 is partly open (Figure 3a). Therefore, POTRA can regulate\ngate dynamics, and may drive the β1-β3 switch between gate-open and gate-\nclosed conformations.\nWe are unsure why different OMPs have retained different POTRA domains. This may be\nattributed to the substrate pool, which is larger for BamA with its five POTRA domains (Biochim\nBiophys Acta Biomembr 2017 1859(12):2467-2482). Another possible reason could be the\nexposure of bacterial insertases to harsher external environments, wherein POTRA may help\nkeep the BamA lumen “closed” to nonspecific entry of molecules from the external environment.\nThe role of POTRA domain in both bacteria and mitochondria is contrasting. In bacteria, it\nassists in precursor binding, whereas it helps precursor release in mitochondria (Mol Biol Cell\n2011 22(16):2823-33). We have briefly mentioned this on page 3, lines 9-11:\nSam50, the core SAM component, is a 16-stranded mOMP insertase, with one N-\nterminal POlypeptide TRansport Associated (POTRA) domain located in the IMS\nthat facilitates precursor release (Figure 1a).\nMinor:\n1. Page 4, lines 15, 31, 32, and 36, It is not clear about the difference between the “path-\nindependent assembly” and the “path-dependent assembly”. The authors should add some\ndefinitions of the two types of assembly.\nResponse page 5\nAuthor response: We apologize for not defining the terminologies. By path-independent\nfolding, we refer to reversible systems that display no hysteresis. Here, the folding and unfolding\nequilibria overlay, and allow for accurate measurements of thermodynamic parameters.\nOne simple difference is:\nPath-independent folding: |Folding free energy| = |Unfolding free energy|\nPath-dependent folding: |Folding free energy| ≠ |Unfolding free energy|\nWe have now removed any mention of “path-dependent folding” in our revised manuscript.\nAdditionally, we define “path-independent folding” as “path-independent reversible folding (no\nhysteresis)” on page 4, lines 33-35:\n[…] are major determinants15 of thermodynamic stability, path-independent\nreversible folding (no hysteresis), and stepwise β-barrel assembly.\n2. Fig 1g, what’s the difference between the control and the FL? It seems like the same\nconstruct but with a different phenotype in some situations.\nAuthor response: The strain we use here is Saccharomyces cerevisiae YPH499 with the\nchromosomal copy of SAM50 deleted (YPH499 sam50::ade2 [Yep352-MET25pr-Sam50-\nCYC1t]; MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1). In this\nstrain, we have the following:\nControl: YPH499 sam50::ade2 strain transformed with yeast episomal plasmid\nYEpSAM50FL (expressing SAM50FL constitutively).\nFL: Complementation of Control (YPH499 sam50::ade2 with YEpSAM50FL) with\npFL39-SAM50FL (expressing full-length SAM50 constitutively), followed by curing\nof YEpSAM50FL.\nΔ120: Complementation of Control (YPH499 sam50::ade2 with YEpSAM50FL)\nwith pFL39-SAM50Δ120 (expressing POTRA-less SAM50 constitutively), followed\nby curing of YEpSAM50FL.\nBoth control and FL are similar, but in FL, Sam50FL is expressed from the pFL39 vector,\nallowing us to directly assess vector-specific phenotypes, and also compare growth of FL with\nΔ120. The details are provided in the SI file.\n3. Fig 1f, the authors should include a negative control such as a liposome without protein.\nAuthor response: Electrophysiology experiments are carried out by painting a planar bilayer\nmembrane across a 150 μm aperture of a polysulfone cup. Since they do not involve a\nliposome, these experiments do not have a negative control. Here, in the absence of a “hole” in\nthe membrane to allow ion movement, the current remains zero at any applied voltage.\n4. Figure 1c. The author did not label the band, which would confuse them.\nAuthor response: We have now labeled the band as Δ120, and added an arrowhead to\nindicate it.\nAdditionally, the accumulation of the ~27 kDa species cannot be observed clearly (lane 5).\nAuthor response: This species accumulates only upon prolonged incubation with proteinase K.\nWe provide this gel image in Figure S1b.\nFurthermore, the folded Sam50 should not be digested by PK without PMSF (lane 6).\nAuthor response: In lane 6, the folded Sam50Δ120 is exposed to proteinase K for the duration of\nthe experiments (20 min), and the gel running time (1 h). All β-barrels are susceptible to\nproteinase K upon prolonged exposure. Here, extramembranous regions (loops, turns) are\ndigested by proteinase K. In turn, this destabilizes the barrel, and the membrane-embedded\ntransmembrane peptide segments are no longer detected on SDS-PAGE.\nResponse page 6\n5. Figure 3b. This MDS should be further confirmed by a single molecular experiment, for\ninstance, smFRET.\nAuthor response: In Figure 3b, we are presenting the change in distance between P228 Cα of\nturn 3 (T3) and the β-barrel midplane (W240 Cα) to highlight the dynamicity of T3. As we are not\nmeasuring the interaction, but the dynamicity that occurs in a few nanoseconds, the latter will\nnot be captured accurately using routine smFRET experiments. Besides, labeling T3 with a\nbulky fluorophore will alter its dynamicity and provide inaccurate read-outs. Therefore, MD\nsimulation appears to currently be the best approach to map these nanosecond dynamics.\nHowever, this is an excellent suggestion, and we are currently exploring the use of smFRET to\ncapture protein-protein interactions and slow association dynamics involving mOMPs.\n6. Page 9 line 11, are the conserved residues in BamAEc or other beta-OMPs also involved in\ntheir folding regulation? The authors should add discussion here.\nAuthor response: We have not come across any study which reports the stepwise folding\nevents of BamAEc or other mOMPs. Indeed, we are the first to present such a finding. Hence,\nwe are unable to make this comparison at this point.\n7. Figure 3a, the results relevant to the all-atom MDS of Sam50120 and full-length Sam50 are\nnot clear. The author should figure out which structure represents the Sam50120, as well as\nthe full-length Sam50.\nAuthor response: Agreed. We have now labelled FL and Δ120 in Figure 3a. We have also\nseparated the FEL for FL as Figure 3d, and presented the FEL for Δ120 and its protein variants\nas Figure 3e. We are sure that this provides more clarity to the images.\n8. In Figures 2 and 4, the author should label the number of the beta-strand for a better\nunderstanding of readers.\nAuthor response: Agreed. We have now added the strand number.\n9. Page 22 line 19, the “red diamond” may be the “red circles”.\nAuthor response: We apologize for the oversight. This is now corrected.\n10. Page 6, lines 36-39, it’s difficult for readers to understand how the data in Figure 4a support\nthe description: “a parallel mechanism for Sam50Δ120 assembly in PC: PE membranes”. More\ndetailed interpretations of the data are suggested here.\nAuthor response: We understand that the text was very technical, and needed some\nexplanation. We observe at least two rates in folding and two rates in the unfolding kinetics.\nThese rates can correspond to one of the two mechanisms:\nMechanism 1:\nUnfolded  Transition state 1  Transition state 2  Folded\nMechanism 2:\nUnfolded  Transition state 1  Folded\nUnfolded  Transition state 2  Folded\nMechanism 1 follows a sequential reaction, whereas, Mechanism 2 follows a parallel reaction.\nTo identify which mechanism Sam50 follows, we used interrupted (un)folding measurements at\ndifferent aging times (t ). Here, we allow the protein to fold only “partially”, and then measure\nage\nthe kinetics of the process. If we get only one rate, that represents Mechanism 1. If we still get\ntwo rates, then it confirms Mechanism 2.\nWe have now provided a detailed explanation of this process in Figure S21, and simplified the\nmanuscript text as below (page 6, lines 27-32):\nResponse page 7\nWe observe two kinetic rates, which can correspond to sequential or parallel\npathways. Our interrupted (un)folding measurements over different aging times\n(t ), and [GdnHCl], provide refolding and re-unfolding rates that match the\nage\ndirect (un)folding measurements (Figures 4a, S20-S21). This supports a parallel\nassembly mechanism for Sam50 in PC:PE membranes. The re(un)folding\namplitudes at each rate show no [GdnHCl] dependence, further supporting\nparallel assembly pathways.\n11. Page 7, lines 3-20. It is also difficult to understand the relationship among the “multiple\npathways of folding”, “parallel pathway”, and the “TS1 and TS2”. The authors should provide\nmore accurate interpretations for this part.\nAuthor response: We apologize for the confusion. We have now simplified the manuscript text.\nWe have only used “parallel pathways” throughout the text. TS1 and TS2 are mentioned only\ntwice in the Results, with the necessary description. The phrase “multiple pathways of folding” is\nnow replaced as below (page 6, lines 33-35):\nThe measured differences in ∆G0,WT and ∆G 0,WT arise from structural and bond\nF,eq kin\nrearrangements in the TS, and the multiple (un)folding rates.\nResponse page 8\nReviewer #2 (Remarks to the Author):\nReferee report for Ravi et al: In this paper, the authors present a series of biophysical and\nbiochemical assays and simulations characterizing the folding and stability of the Sam50 protein\nin model membranes. They further perform in vivo assays in yeast to assess lethal sets of\nresidue mutations. The introduction establishes that this mitochondrial membrane protein is\nessential for cells and notes that defects in its function contribute to mitochondrial disorders.\nOverall, the authors have collected large datasets to comprehensively assess single-point\nresidue mutations (none of which are lethal), some ‘multipoint’ mutations, as well as assays that\nidentify folding transition states. While these datasets will be useful to researchers studying the\nprotein Sam50, the paper is extremely technical and does not establish a clear or coherent story\nabout how all of this data is broadly useful or insightful to non-experts or a broad audience in\nbiology or biophysics, or how it will be useful for therapeutics or disease. A persistent issue is\nthe use of words that are not standard in biophysics or are misused, along with statements that\ndo not make sense. There are many aspects of the system that are not clear even after reading\nthe paper several times. For example, is the folding ever studied in the membrane in the\npresence of the SAM chaperone? Is it the Sam50 dimer (Sam50b and Sam50a)? Or just one\nsubunit? A major issue is that the collected results are not integrated together. The MD\nsimulations in particular are almost completely divorced from the study of folding of the protein,\nas they only compare energy landscapes of structures under mutations, showing rather minor\neffects. Many of the figures do not support strong conclusions (Fig 3 and 4), and I am not sure if\nFig 5 is speculation or data.\nAuthor response: We thank the Reviewer for assessing our initial submission. The Reviewer\nhas indicated that the paper is too technical and very difficult to understand. We want our\nfindings and our manuscript to reach the broadest audience of Nature Communications.\nTherefore, and to ensure that our work is appreciated by both biologist and chemists, we have\nthoroughly rewritten the entire manuscript to make it a storyline that portrays our very interesting\nfindings in a manner that will appeal to the broad audience of both biologists and chemists, and\ninitiate novel research on developing therapeutics. Unlike in vivo experiments or cryoEM\nstructures that provide limited information, we have specifically chosen the study of Sam50\nmonomer in membranes of defined composition to map the stepwise events that lead to folding\nof this protein. We have additionally integrated residues vital for the folding process with\nresidues that contribute to stability and dynamicity of this β-barrel, and assigned a functional\nsignificance to these residues with our in vivo data. Each specific concern of the reviewer has\nbeen thoroughly addressed in the subsequent pages. Overall, we are confident that the\nReviewer will find the revised submission suitable for publication.\nGeneral issues:\nThe abstract is already very difficult to parse.\nAuthor response: We thank the reviewer for the assessment. We have now rewritten the\nabstract and simplified it considerably.\nWhat does ‘regulators’ mean?\nAuthor response: We refer to residues that play a role in the folding or stability or function of\nSam50, as its molecular regulators. These residues act as control points.\nMDS is not a standard acronym, even MD would be better.\nAuthor response: MDS is now rephrased to MD simulations.\n165 variants of Sam50 is actually 165 mutations to the Sam50b subunit? Multipoint is not a\nfamiliar or standard word in biophysics: at multiple residues in the sequence. I am not sure why\nthe word mutation is rarely used.\nResponse page 9\nAuthor response: Sam50a and Sam50b are identical subunits, wherein Sam50 forms a\nhomodimer. They are demarcated as Sam50a and Sam50b, upon binding to Sam35 and\nSam37, respectively. In our in vitro experiments, we only study one Sam50 molecule.\nMultipoint mutation is now changed to multiple mutations. We have retained “multipoint” in some\nplaces where required, as this has been used previously in the literature as well (Nat Struct Mol\nBiol 2023 30(2):176-187).\nThe word “mutation” refers to what happens in the DNA. This results in a difference in the\nprotein sequence. Proteins, by definition, cannot be “mutated”; they can only be synthesized\nwith an “altered” sequence. However, the use of this correct terminology has now become a\nrarity in the scientific literature. Nevertheless, in keeping with the current trends, we have\nintroduced the word “mutation” in some instances, wherever appropriate. In others, where we\nrefer to the protein, we have used the word “variant”.\n‘This metastable structure’? Which metastable structure is this referring to?\nAuthor response: We were defining Sam50 as less stable than other 16-stranded OMPs.\nHowever, since this terminology was confusing, we have now removed the word metastable\nfrom the manuscript.\nAnd again, what are ‘microscopic regulators’?\nAuthor response: We were referring to amino acid side chains as regulators. Since term\n“microscopic regulators” was confusing, we have now removed it from the manuscript.\nWhat is conformational buffering?\nAuthor response: We use the term conformational buffering/ conformational dynamicity to\ndescribe the conformational switch from “gate closed” to “gate open” state and vice versa in\nSam50. Since term “conformational buffering” was confusing, we have now removed it from the\nmanuscript.\nWhat are structure-function handles?\nAuthor response: We found a specific localization of residues that are intrinsically destabilizing\nat β4-β6. We also find β4-β6 as important for function of Sam50. Hence, we called this as\nstructure-function handles. Since this term was confusing, we have now removed it from the\nmanuscript.\nIn short, the authors do not seem to be entirely clear what they are trying to say, and have\nleaned on broad generalizations that muddy what approach they have taken and what they\nhave learned. The word regulators needs to be replaced with something specific, like residues,\nsecondary structures, or something that reflects what they tested and learned.\nAuthor response: When we refer to word regulators, it refers to the amino acid that controls\nthe stability, folding, or function. We have now replaced it with residues, structures (specifying\nthe strands/ residues), or removed the phrase entirely. Other changes, as suggested by the\nReviewer, have already been incorporated, and the manuscript now reports specifics of what we\nhave identified and obtained from our experiments.\nThese sentences contain more information: “We show that Sam50 folds through multipoint\nnucleation and N-terminal structuring, whereas C-terminal frustrated folding forms kinetically-\ntrapped intermediates. This unexpected folding correlates mechanistically with SAM-assisted in\nvivo assembly”. However they are still confusing. When does the C-terminal folding\noccur, do they see this in the membrane? The fact that this is even undergoing folding in the\nmembrane is not clear, nor is it clear that they look at only one subunit, or that the SAM\nmachinery is known to assist in protein folding within membranes.\nResponse page 10\nAuthor response: We thank the reviewers for these questions. We believe that there may be\nsome confusion here regarding mOMPs, SAM organization, and Sam50 folding. Here are our\nsuccinct responses:\na) Our experiments show that the C-terminal strands form non-native interactions, and\ntherefore, these strands do not assemble early during the folding process (Figure 5, grey\nshaded area). We have also explained how this is overcome in vivo – with SAM\nassistance.\nb) We have verified that Sam50 indeed folds in vitro without SAM assistance. We used four\ndifferent experimental approaches that could be tested simultaneously on Sam50-FL,\nSam50-Δ120, or any of the Ala variants:\n1. Resistance to proteolysis by a robust non-specific protease such as proteinase K\n(Figure 1c). Here, an unfolded/ membrane-adsorbed/ partially folded protein is\nrapidly digested by proteinase K (shown in the same gel).\n2. β-Rich secondary structure content confirmed using far-UV circular dichroism\nspectropolarimetry (Figure 1d). The structural content is comparable to reported\nellipticity values for another 16-stranded bacterial β-barrel.\n3. Change in Trp fluorescence intensity (2-fold increase) and emission maxima (10-nm\nblue-shifted emission) (Figure 1d, inset).\n4. Single-channel electrophysiology of these artificially folded samples shows gating\ncharacteristics that match previous reports which studied reconstituted SAM complex\n(Figure 1f).\nNo other study offers this extent of confirmation that a membrane protein is indeed\nfolded.\nc) It is not clear what the Reviewer means by “one subunit”. If they are referring to Sam50a\nand Sam50b, we have already clarified that both are identical polypeptides, and the\nnomenclature is simply because of the Sam35/37 that they bind to.\nd) Previous studies have clearly established that mOMPs can assemble without SAM\nassistance. We draw the Reviewer’s attention to the SAM-independent unassisted\nfolding reported for several other mitochondrial OMPs (including yeast Por1, mouse\nVDAC1, and the three human VDACs 1/2/3), and have been demonstrated in artificial\ndetergents, bicelles, and vesicles.\nThe authors describe the structure as “metastable” throughout the text. This has a strict\nthermodynamic meaning: this structure is not the free energy minimum, but can persist for some\ntime range because it has a barrier separating it from the free energy minimum. This is therefore\nnot the most favorable equilibrium structure. When do they show this? It appears to be asserted\nwithout support. They seem to be abusing the word metastable, or at least haven’t actually\nprovided evidence in the text of the metastability. To the contrary, that the protein folds to a\nfunctional state on its own despite many mutations seems to indicate overall stability.\nAuthor response: A metastable structure is not necessarily a transient state that persists\nduring protein folding. In fact, a metastable structure can be an equilibrium structure, and we are\nnot the first to discuss the metastable nature of a protein. The Muller lab has indeed measured\nthe metastable nature of BamA (Structure 2018 26(7):987-996.e3.). Various factors like\nmembrane composition, extracellular loops and POTRA domains contribute to the metastable\nnature of a protein, and increase its dynamic nature for superior function (e.g., the dynamicity\nrequired during substrate assembly). Another study that used biophysical tools identified the\nmetastable nature of the β-barrel OMP Ail (J Am Chem Soc 2022 144(4):1545-1555). Hence, we\ndisagree with the Reviewer’s comment that a metastable state is unfavorable. That being said,\nwe have changed metastable to less stable or moderately stable throughout the manuscript.\nResponse page 11\nThe MD simulation results are largely divorced from the rest of the study. Despite having\nextensive residue-level experimental data, the simulations are not directly compared to the\nexperiments in any way. The main conclusion is that the free energy landscape becomes wider\nas a function of the RMSD, but they already showed that that protein has a wider RMSD\nbecause it has an extra domain, so it will automatically sample more values. These are not\ninsightful plots about the function. They have a proposed folding mechanism and it is not\ninterrogated using the atomistic simulations.\nAuthor response: We disagree with the comments, and particularly to the use of the word\n“divorced”. The FEL is not our key point of discussion at all. With MD simulations, we show how\nresidues that are stabilizing or destabilizing are affecting the energy states and dynamicity of\nfolded Sam50. We demonstrate how the presence of POTRA leads to a compact TMD – this is\nthe first report of this property of POTRA. We additionally demonstrate how allosteric regulation\nis effected between mid-barrel residues and T3. We show how substitutions at specific residues\ncan alter conformational sampling in Sam50. The proposed folding mechanism derived from\nkinetics measurements is distinct from the dynamics we obtain for the post-folded structure in\nour MD simulations, and there is nothing to interrogate in this. Therefore, we find it logical to\ncompare only the equilibrium thermodynamics with the MD simulations.\nThe authors refer to a ‘kinetic free energy’ (P7L4) and ‘energetic contributions from both kinetic\nand thermodynamic components’ (P10L31). This is misleading. Free energies are by definition\nequilibrium measurements that have no kinetic component. If they extracted them from\nexperiments that also measured kinetics, that is fine, but don’t assign this confusing language to\na free energy.\nAuthor response: Yes, by and large, free energies are equilibrium measurements. However,\nfor membrane proteins, it has been demonstrated through several studies that kinetic\ncomponents also contribute to the measured free energy. A seminal study demonstrated this\nusing diacylglycerol kinase (J Am Chem Soc 2013 135(40):15183-90). The reason kinetic\nstability plays a major role is because these proteins are “locked” or “kinetically trapped” once\nfolded in the membrane. Contributions from kinetic stability, along with the conventional\nthermodynamic stability, have now been accepted for membrane proteins, and are also being\nconsidered for soluble proteins. That being said, we have now simplified the sentence (page 10,\nlines 5-8) and cited the relevant paper:\nThis can arise from contributions of both kinetic and thermodynamic stability, as\nseen in bacterial OMPs.24 Indeed, long-chain lipids increase Sam50 stability but\nwith hysteresis (Figure S2), suggesting a lipid-mediated kinetically trapped\nSam50 β-barrel in vivo.\nThe following specific parts in the manuscript require further clarification and/or restructuring:\nOn page 3 it is mentioned that the SAM complex helps to fold Sam50. However, all of the\nresults seem to be Sam50 folding by itself in the membrane, rather than folding with the\nassistance of the SAM chaperone activity.\nAuthor response: In the cell, the SAM complex assists Sam50 folding. However, we have\ndemonstrated here that Sam50 shows unassisted (SAM-independent) assembly. SAM-\nindependent unassisted folding of other mitochondrial OMPs (including yeast Por1, mouse\nVDAC1, and the three human VDACs 1/2/3) have been demonstrated in artificial detergents,\nbicelles, and vesicles. Hence, our finding that Sam50 can fold without the help of SAM/BAM,\nwhile not unexpected, has not been shown before.\nFigure 4: I am not sure how to interpret these plots, they seem more like supplementary\ninformation. There are a series of points and lines, and what I am supposed to expect vs what\nResponse page 12\nthey see is not clear from the plot. Comparison of (d) and (e) requires staring at these\nsequences and picking out variations, none of which are highlighted and made clear. Yes there\nare differences between them, what do I learn from this?\nAuthor response: Figure 4a-c shows the chevron plots obtained from the kinetics\nmeasurements. All those engaged in studying protein folding know how important it is, as it\nforms the backbone of their study, and include these plots in their manuscripts. Figure 4a-b\ndemonstrates that Sam50 follows parallel folding pathways. Figure 4c shows representative\nunfolding rates for a few protein variants, which are compared with the WT, to demonstrate the\ndifferences/ similarities. Figure 4d-e is now presented as Figure 5a-b. These are two-\ndimensional topology diagrams that map the results from the -value analysis (another favorite\nof all those who study protein folding), and provide a direct read-out of which part of the protein\nfolds first. To ensure that the information is presented in a simplified manner for non-experts,\nand in keeping with the Reviewer’s suggestion, we have now highlighted three zones in the\ntopology maps, which fold (zones i, ii) or form non-native contacts (zone iii). We thank the\nReviewer for pointing it out, as this change has now made it convenient for the reader to\nunderstand the topology maps.\nFigure 5: Are these results or speculation? Where did the Tims come from? These proteins were\nnever mentioned and were not included in the experiments.\nAuthor response: The small Tim chaperones were included in the model we were proposing,\nwherein we had integrated our observations with what is believed to occur in the mitochondrion.\nWe have now removed Tim/SAM from this proposed model, and illustrated only the mechanism\nwe obtained from the unassisted folding of Sam50 exclusively from our study.\nPage 3: what do the superscripts mean on the names of proteins? Some are Sam50, one is\nSAM, why?\nAuthor response: We understand that this nomenclature has caused some confusion. We\nhave now simplified it as follows:\nSam50: the core β-barrel protein we are studying here. For simplicity, we have\nreferred to Sam50Δ120 as Sam50 only.\nSam50FL versus Sam50Δ120: Wherever demarcation between the full-length and\nPOTRA-free variant is required, we have used these nomenclatures.\nSAM: Sorting and Assembly Machinery (the entire complex found in vivo).\nP4 L38: Protein folding is bimolecular? What does this mean? Bimolecular reactions typically\nrefer to two species at well-mixed concentrations binding to one another through space. Folding\nis a polymer dynamics problem. At no point in the SI does it show a bimolecular treatment of the\nobserved dynamics. Sums of exponentials are not indicative of a bimolecular reaction (which\nhas units of M-1s-1 for rates).\nAuthor response: What we meant here as a “bimolecular reaction” for a membrane protein is\nthe unfolded protein (molecule 1) and the LUVs (molecule 2). To retain simplicity and avoid\njargons, we have removed any mention of a bimolecular process in the manuscript.\nP5 L5: Surface area in units of kcal /mol /M, this is completely unfamiliar to non-experts.\nAuthor response: What we measure here is the total change in the protein’s accessible\nsurface area as a function of a perturbant. This is an empirical parameter, denoted as M ,\nand\nis also referred to as folding cooperativity. The conventional unit for change in accessible\nsurface area is Å2. However, when this empirical parameter measures the dependence of the\nchange in free energy on the perturbant concentration upon protein folding or unfolding, it is\ngiven the unit of kcal/mol-M. To avoid any confusion, we now refer to M as “folding\neq\ncooperativity”.\nResponse page 13\nFig 3a overlaid structures are very difficult to understand. What are we supposed to be seeing\nhere?\nAuthor response: This figure is an overlay of Sam50FL and Sam50Δ120, color-coded based on\nthe pairwise RMSD values. Here we show how the compaction of the TMD, and a reduction in\nthe strand tilt angle (with respect to the bilayer normal), is seen when POTRA is present. We\nhave now labeled the figure to distinguish both transmembrane domains.\nFig 3d: what do we learn from these plots? It is unsurprising that FE landscape is broader with\nrespect to RMSD when there’s a large domain added that increases the RMSD.\nAuthor response: Yes, RMSD increases with the POTRA domain, which is the reason we see\na broader FEL for full-length Sam50. Now we have split this panel to Figure 3d (only FL-Sam50\nwith the POTRA) and Figure 3e (that only compares the POTRA-deleted Sam50 and its\nvariants). What we learn is that a broader FEL in FL-Sam50 is because of contributions from the\nRMSD. Differences in the FEL of Δ120 and its protein variants are from contributions of both\nRMSD and RMSF.\nFig 3d D120 landscape: are those two bumps supposed to be the metastable and stable states?\nAuthor response: These are the energy states sampled by the protein, and do not correspond\nto the metastable and stable states. We also emphasize here that Sam50Δ120 is a metastable\nstructure, which is a thermodynamic description. Nowhere have we mentioned that Sam50Δ120\nshows both “metastable” and “stable” states.\nFig 3c: Are these curves statistically significantly different? Error shading?\nAuthor response: We only interpret T3 and L4, which show clear difference in the RMSF. The\nshading is for the β-strands, which we had mentioned in the figure legend. We have now\nadditionally labeled it in the figure itself to avoid any confusion.\nStatements that are unclear and are not supported by data:\n'Beta barrel stability allosterically regulates protein dynamics'? How does ‘stability’ allosterically\nregulate dynamics? I have no idea what this is trying to say.\nAuthor response: Proteins are not rigid entities. It is widely known that in order to become\nhighly dynamic, a protein must have lower stability, so as to provide the necessary flexibility\nessential for its function. Now, this can be advantageous for a protein, whereby, in increasing its\noverall scaffold dynamicity, it lowers its overall scaffold rigidity. As a matter of fact, metamorphic\nproteins work on this very concept, and, many studies have linked low stability of the protein\nwith its high dynamicity. For example, with membrane proteins, the attachment and invasion\nlocus (Ail) of Yersinia pestis has high percent identity in sequence and structure with E. coli\nOmpX. But, both proteins differ hugely in their thermodynamic stability. OmpX is much more\nstable than Ail, and as a consequence, it has lost its function of “adhesion” in the course of\nevolution. On the contrary, Ail, with much lower stability, is a key reason for the pathogenic\nnature of Yersinia pestis (J Am Chem Soc 2022 144(4):1545-1555.).\nSpecifically for Sam50, this “flexible” nature is vital for the protein to switch between gate-closed\nand gate-open states for its function. Hence, we show that the less stable nature of Sam50 is\nimportant for its “dynamic” gating function.\nP6 L22: “Our simulations establish how intrinsically destabilizing residues modulate signal\npropagation across the membrane, with zonal changes (cytosolic Sam35/37 binding, N-terminal\ngating, substrate interaction) allosterically propagating through the β-barrel, to alter IMS\ndynamics and regulate Sam50 function”. What are zonal changes? Again the word regulate is\nResponse page 14\nused but it is nonspecific, regulate how? How am I to interpret ‘modulate signal propagation’?\nWhat signal?\nAuthor response: We understand this might be a little confusing. Our MD simulations show\nthat changing the TMD residues at strands β7 and β15, can alter the dynamicity of the\nextramembranous loop L6, and turn T3. L6 is involved in binding with Sam35/37 and T3 can\ninteract with the single POTRA of Sam50. What we propose based on our MD simulation results\nis that changes in the TMD zone can affect extramembranous regions. Changes which occur in\none region (zone) of the protein but have an effect in another distant region (zone) of the protein\nare considered allosteric changes. Allostery is not restricted to protein-ligand interactions, and is\nseen in intra-protein and protein-protein interactions also (two excellent reviews in this subject: J\nMol Biol 2008 378(1):1-11; Trends Biochem Sci 2023 48(4):375-390). In β-barrel membrane\nproteins, allosteric regulation at a mortise-and-tenon motif (e.g., OmpX Y80-G112; PLoS One\n2017 12(8):e0182016) propagates the signal for correct β-barrel folding and prevents\nmisfolding. A similar mortise-and-tenon motif in the C-terminus of autotransporters modulates\ntranslocation of the N-terminal passenger domain to the bacterial surface, offering the functional\ncharacteristics (Nat Commun 2014 5:4239). That being said, given how such elaborate\nexplanations cannot be offered in the manuscript, we have now simplified these sentences and\nrephrased it as follows (page 6, lines 5-10):\nOur simulations reveal that this increase in dynamicity of T3 of 18 residues\n(unlike longer loops L6 (41 residues) and L4 (31 residues)), is due to A240 (on β7)\nand A455 (on β15) causing an allosteric effect on T3. Hence, the midplane W240\n(β7) and F455 (β15) anchor the TMD, help form structured extramembranous\nsegments, and are vital for Sam50 structural integrity (Figures S12-S15). The loss\nin membrane anchoring explains the destabilization we observe when W240 or F455\nis replaced (see Figure 2d).\nWhy do the authors fit to a triple exponential for folding and a double exponential for unfolding?\nWhere did this choice for the number of rates come from?\nAuthor response: As is well-established in the literature, the choice of an exponential depends\non the quality of the fit we obtain. One must avoid problems with both over-fitting and under-\nfitting. We assessed this based on the quality of the fits, which we provide in Figures S17 and\nS18. The residuals are a measure of fit quality. The minimum exponent that provided acceptable\nresiduals for folding was a triple exponential rise function, and that for unfolding was a double\nexponential decay function. Hence, we used these appropriate equations.\nS19: Why do the kinetics and equilibrium measurements overlay so well (“excellent overlay”)\nwhen at the same time,\n“Sam50Δ120 undergoes slow rearrangement (possibly through multiple pathways of near-equal populations), which cannot\nbe captured in the rapid kinetics measurements, but is evident in the equilibrium titrations.”?\nAuthor response: We wish to clarify this. What we present in Figure S19 is the end-state\nfluorescence, and this is the read-out we obtain at the end of the reaction. We draw the\nattention of the Reviewer to the data points that are not available in the kinetics (symbol:\ntriangles), and these points are missing because we are unable to measure the rates at the\nintermediate GdnHCl concentrations. The slow rearrangement we see in Sam50Δ120 is at these\nintermediate GdnHCl concentrations only. These slow rearrangements take min-h, while the\nkinetics is captured in seconds. The overlay remains only in those regions wherein the reaction\nis completed fast, and is in line with what we claim here.\nResponse page 15\nReviewer #3 (Remarks to the Author):\nThis manuscript by Ravi et al. presents a detailed and multifaceted investigation into the folding\nmechanism, stability, and functional regulation of the mitochondrial outer membrane protein\nSam50. By integrating thermodynamic profiling, ultrafast folding kinetics, molecular dynamics\nsimulations, and in vivo functional assays, the authors provide detailed insights into the\nfolding landscape and structure-function relationships of Sam50.This study addresses a significant\ngap in our understanding of mitochondrial β-barrel assembly. Major Strengths include the\ncomprehensive approach combining experimental and computational tools. Also, the mapping of\nthermodynamically (de)stabilizing hotspots and their in vivo validation enhances the\ntranslational relevance of the work. I have a few points that need to be addressed before\nconsidering publication.\nAuthor response: We thank the Reviewer for appreciating our work and for sharing the\nsuggestions. We have revised our manuscript to incorporate all the suggested changes, and we\nare positive that the Reviewer will find the revised submission suitable for publication.\nThe folding studies were performed in a PC:PE (90:10) lipid system. While this is experimentally\ntractable, it doesn't fully mimic the native mitochondrial outer membrane environment. The\nauthors mention the use of \"native-like\" conditions, but this lipid mixture is distinct from actual\nmitochondrial membranes. The manuscript would benefit from a discussion on how the choice\nof lipid composition may influence Sam50 folding energetics and the generalizability of the\nfindings to in vivo conditions.\nAuthor response: We agree that PC:PE is not a non-native system, and we have now\nremoved this phrase to avoid any confusion (page 4, line 10). Our choice of lipids was limited by\nthose systems which supported path-independent folding of Sam50. We also welcome the\nsuggestion to discuss the role lipids play in Sam50 energetics, particularly in vivo. Optimization\nof Sam50Δ120 folding in different lipids gave us insights into the effect of lipids on stability.\nLonger chain lipids increase protein stability (Figure S2). We have discussed this briefly on page 10\nlines 6-8:\nIndeed, long-chain lipids increase Sam50 stability but with hysteresis (Figure S2),\nsuggesting a lipid-mediated kinetically trapped Sam50 β-barrel in vivo.\nThe manuscript notes the potential for structure-based peptidotherapeutic development.\nHowever, this important point is not developed in detail. The authors should elaborate on which\nstructural or energetic features of Sam50 could be rationally targeted for therapeutic design and\nhow their findings might inform future efforts. As it stands, the therapeutic implications are\nspeculative without further context.\nAuthor response: We have identified β4-β6, Y195 (β5), W240 (β7), K341 (β11), D385 (β12), V420\n(β13), F455 (β15), G443 (β14), P462 (β15), and T3, as hotspots of Sam50 that may potentially\nserve as drug targets. In keeping with your comment, while also accommodating the concern of\nReviewer 1, we have now removed the mention of peptidotherapeutics in the Abstract, and\nprovided the specific details in the last section of the Discussion (page 11, lines 19-22),\nas\nbelow:\nOur findings help understand how evolution attained structural fine-tuning\nthrough key structures and residues (including β4-β6, Y195 (β5), W240 (β7), K341\n(β11), D385 (β12), V420 (β13), F455 (β15), G443 (β14), P462 (β15), and T3), which\nalso direct function, stability, and regulation in vivo.\nWe have also rephrased the Conclusion (page 11, lines 35-38), as below:\nThe identification of residues tailored for stability and functional efficacy for vital\nproteins, such as what we report for Sam50, can unveil mechanistic correlations\nResponse page 16\nof mOMPs under both health and disease states, furthering the design of possible\ntherapeutic molecules.\nThe figure captions are too long and include large amounts of methodological and interpretive\ninformation, which would be better placed in the main text. The paper would benefit from\nconcise captions and improved integration of the relevant details into the Results and\nDiscussion sections.\nAuthor response: We welcome this suggestion, and we have shortened the legends of Figures\n1-5. We have retained some explanation in Figure 6, which is the schematic, so that readers\ncan immediately relate the illustration with the information.\nMinor points:\nPage 5: I didn't understand the units of surface area (\"3.01 kcal/mol*M\"). Can the authors\nclarify?\nAuthor response: What we measure here is the total change in the protein’s accessible\nsurface area as a function of a perturbant. This is an empirical parameter, denoted as M ,\nand\nis also referred to as folding cooperativity. The conventional unit for change in accessible\nsurface area is Å2. However, when this empirical parameter measures the dependence of the\nchange in free energy on the perturbant concentration upon protein folding or unfolding, it is\ngiven the unit of kcal/mol-M. To avoid any confusion, we now refer to M as “folding\neq\ncooperativity”.\nPage 5: \"other transmembrane Phe(18), Tyr(6), and Trp(3), do not stabilize the...\" - maybe say\nresidues after Phe, Tyr, and Trp?\nAuthor response: We have now simplified it to (page 5, lines 20-21):\nOther transmembrane aromatic residues do not alter Sam50 stability (Table S2),\nsuggesting a location-specific contribution of W240 and F455.\nPage 12: \"evisage\" - should be envision\nAuthor response: Rephrased.\nPage 12: \"drug designing\" - should be design\nAuthor response: Rephrased.\nResponse page 17\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nAuthor response: We thank this early career researcher for her/his time, and we appreciate\ntheir effort. We also wish her/him good luck in their career.\nResponse page 18\nManuscript NCOMMS-25-06253A-Z\nTitle: Linking multipoint β-barrel folding with stability and regulation in the mitochondrial outer\nmembrane protein Sam50\nReviewers' comments and Authors' responses\nReviewer #1 (Remarks to the Author):\nRavi et al have addressed a part of my concerns. This study offers insights into how Sam50’s\nfolding process is regulated by its β-strands and loops. The additional revisions have\nstrengthened the manuscript; however, despite the authors' efforts to address the reviewers'\ncomments, some aspects still require further clarification. Given that it is the first time showing\nthat Sam50 can fold successfully in membranes in an in vitro system, without assistance from\nthe SAM complex, the authors should provide more compelling evidence showing that this\nspontaneous insertion system works (see below). Moreover, they should better interpret\nwhether this spontaneous insertion process could occur in the mitochondrial outer membrane\nunder physiological conditions.\nResponse: We thank the reviewer for appreciating our efforts and sharing the suggestions. The\nreviewer highlights that this is the first study to report the unassisted folding of Sam50 without\nthe assistance of the SAM complex. However, Paschen et al., Nature, 2003 (cited by the\nreviewer below) have already demonstrated the unassisted folding of Sam50 (it was initially\ncalled Tob55). They expressed\nNeurospora crassa Sam50 (Tob55) in E.\ncoli as inclusion bodies, extracted and\nsuccessfully folded the protein in vitro.\nWe have followed the same method with\nSaccharomyces Sam50.\nThe Figure 2 of Paschen et al.,\nNature, 2003 is reproduced as-is here\n(left). Panel c shows the step-wise\nincrease in current, panel d has the\noverall channel conductances, and panel\ne has its voltage-dependence. Hence, it\nis well-established that Sam50 can fold\nsuccessfully in membranes in an in vitro\nsystem.\nWe welcome the new\nsuggestions, which we find very helpful,\nand the opportunity to re-revise our work.\nWe have incorporated the necessary\nchanges, and linked our findings (with\nthoughtful consideration) to the in vivo\nprocess.\nPaschen, S., Waizenegger, T., Stan, T. et al. Evolutionary\nconservation of biogenesis of β-barrel membrane proteins. Response page: 1\nNature 426, 862–866 (2003).\nhttps://doi.org/10.1038/nature02208\nMajor:\n1. Although the authors showed four results that tried to prove the Sam501-120 can be\nspontaneously inserted into the artificial membrane, there are also things that need to be\nvalidated.\na)Figure 1c. The folded/unfolded Sam50Δ1–120 cannot be digested by proteinase K in the\npresence of PMSF, since PMSF should fully inhibit PK. As the 27-kDa band appears under\nsimilar conditions in Figure S1b, the authors should show this digestion result in Figure 1c\nwithout PMSF to clarify this point.\nResponse: We believe there is some confusion here, and we apologize for it. To clarify, we first\nadded PK, then incubated the reaction for a defined time, and then added PMSF to arrest the\nreaction. To illustrate this better in Figure 1c, we have now rearranged the annotations above\nthe gel, and added an arrow to indicate the order of addition as PK first and then PMSF.\nAdditionally:\n Shorter incubation times in PK (as shown in Figure 1c) retain the fully folded Sam50 of\n~41 kDa.\n Longer incubation times in PK (as shown in Figure S1b) shows the accumulation of ~27\nkDa band, corresponding to the transmembrane region of folded Sam50 devoid of\nextramembranous loops.\nThe legend of Figure 1c has also been edited accordingly in the revision, to reflect this change,\nas follows (page 18, lines 9-13):\n“(c) Folded Sam50 exhibits protection from proteinase K (PK) digestion. After\nPK addition (+), samples were incubated for the indicated time. Following this,\nPMSF was added to arrest PK. Green arrow: order in which experiment was done.\nOnly reactions not arrested by PMSF addition (#) and the unfolded protein are\nsusceptible to proteolysis.”\nb)Figure 1d. I suggest separating the two datasets in Figure 1d into two individual panels to\nenhance readability.\nResponse: This is now changed as suggested.\nc)Figure 1f. From the perspective of experimental completeness, a protein-free planar bilayer\nmembrane should be included as a negative control in the electrophysiological recordings, even\nif no current is expected. This would help confirm that any observed current is indeed\nattributable to Sam50.\nResponse: We have now added this in Figure 1f.\nd)The electrophysiological experiment is intended to demonstrate that the refolded Sam50 is\nfunctional. However, ScSam50 has not been reported to act as an ion channel. Therefore,\nadditional evidence is needed to confirm that Sam50 was fully folded in the authors’ system.\nAlternative functional assays or structural confirmation would be helpful.\nResponse: We would like to share with the reviewer that it has indeed been demonstrated in\nFigure 2 of Paschen et al., Nature, 2003 (as provided in this response file on page 1) that\nNcSam50 can be extracted from inclusion bodies after expression in E. coli. This study\ndemonstrated the following, which is also in agreement with our results:\nResponse page: 2\n NcSam50 can be successfully folded from urea into membrane mimetics (their Figure\n2a-b), similar to what we show for ScSam50 in our Figure 1b-c.\n NcSam50 thus folded exhibits a characteristic secondary structure content (their Figure\n2b), similar to what we show for ScSam50 in our Figure 1c and several representative\nmutants in Figure S6.\n NcSam50 shows a conductance of 1-4 nS in black lipid membranes (their Figure 2c-d,\nsimilar to what we show for ScSam50 in our Figure 1f.\n NcSam50 displays voltage dependence of channel conductances (their Figure 2e),\nsimilar to what we show for ScSam50 in our Figure 1f.\nIn addition to these similarities with established results, we have nevertheless demonstrated\nthat ScSam50 is folded in our experiments using PK digestion, change in fluorescence profiles,\ntwo-state cooperative folding and unfolding profiles, and characteristic folding and unfolding\nrates that give rise to V-shaped chevron profiles. We have also demonstrated a similar behavior\nfor the 165 mutant variants of this protein. We do not observe aggregation in any of our samples\ndespite prolonged incubation (up to 5 days). Based on all these observations, we are confident\nthat all the Sam50 proteins we characterize in this study are folded. We have also provided\nadditional electrophysiology data in the SI Figure S1e-j.\n2. The folding behavior of β-OMPs in artificial membranes may not reflect their folding in\nmitochondrial outer membranes. Given that Sam50 is essential for yeast viability (Paschen et\nal., Nature, 2003; Kozjak et al., J Biol Chem, 2003; Gentle et al., J Cell Biol, 2004), the authors\nshould discuss whether β-OMPs can spontaneously fold in mitochondria in the absence of the\nSAM complex. Furthermore, the discussion should be expanded to include other β-OMPs\nmentioned in the rebuttal letter, such as Tom40 and VDACs.\nResponse: This is an excellent suggestion. Based on what we find, and based on existing\nreports, we have now added the relevant details in the Discussion, under these sections on\npages 9-10 and 11, respectively:\nMultipoint assembly of Sam50 β-barrel correlates with in vivo molecular\nregulation\nAllostery links extramembranous loop/turn/POTRA dynamics with TMD\nHere, we now compare similarities and possible differences between Sam50 folding in the\nmitochondrion with the steps and intermediates we characterize in our study. We have also\ndiscussed the possible role of POTRA. Structures of VDAC folding intermediates are not yet\nreported. Hence, we have compared our findings on Sam50 with the structural intermediates\ncaptured for Tom40 using cryoEM.\n3. In the rebuttal letter, the authors mention three cryo-EM studies that captured the SAM\ncomplex “in action” — with fully folded Mdm10, fully folded Tom40, and partially folded Tom40.\nHowever, in Figure 6 (panel “2”), only the interaction residues of “SNNP” within the SAM and\nSAM–Mdm10 structures are mapped. The authors should also address: 1) The “SNNP” motif in\nthe two SAM–Tom40 structures. 2) The corresponding residues in the AlphaFold-predicted\nSAM–Porin complex.\nResponse: We have now included these suggestions. We have generated the overlaid\nstructures obtained from SAM with Mdm10 (PDB ID: 7BTY), with Tom40 (PDB ID: 7E4H), and\nResponse page: 3\npartially folded Tom40 complex (PDB ID: 7VKU). These overlaid structures are now shown in\nFigure S33 of the supporting information. We did not add the AlphaFold structure, as the\ninteraction interface is less reliable than cryo-EM for membrane proteins.\nMinor:\n1. Figure 6, the author should use “a, b, c” as icons rather than “1, 2, 3”.\nResponse: Now edited. We have now used “a, b, c” for the subpanels, and Roman numerals for\neach step/substructure.\n2. Throughout the manuscript, replace “mOMP” with the more widely accepted term “β-OMP.\nResponse: Now replaced.\n3. The term “insertase” is used to describe Sam50 in the revised version. However, this term is\ntypically reserved for YidC/Alb3/Oxa1 family proteins, which mediate the insertion and folding of\nα-helical membrane proteins. The authors should reconsider this terminology or provide\njustification for its use in this context.\nResponse: We understand the concern. As suggested by the reviewer, we have rephrased the\nsentences to avoid using the word “insertase” or “chaperone” to describe Sam50.\n4. The role of the POTRA domain should be discussed more thoroughly in the Discussion\nsection.\nResponse: We welcome the suggestion. The function of the Sam50 POTRA domain is not as\ncharacterized as its BamA counterpart in Gram-negative bacteria. Hence, we have now edited\nthe Discussion and mentioned the possible functions of Sam50’s POTRA in our revision. This\ncan be found on page 11, in the third paragraph of the following section:\nAllostery links extramembranous loop/turn/POTRA dynamics with TMD\n5. The English language in the manuscript remains awkward in places and sometimes difficult to\nfollow. Careful proofreading and editing are needed to improve the clarity, flow, and overall\nreadability of the text.\nResponse: We have re-edited sections that appeared difficult to follow. We also used “Nature\nResearch Assistant” to improve the language of the text. We hope the reviewer now finds the\nreadability improved substantially.\nResponse page: 4\nReviewer #2 (Remarks to the Author):\nThe authors have significantly improved the text through rewriting their manuscript, with more\nprecise language, improved integration of the distinct parts into a coherent narrative, and\nimproved figure clarity. Additionally, they have clarified many of the details and confusing\nlanguage. However, we still have issues that we feel should be addressed:\nResponse: We thank the reviewers for their suggestions. We have now addressed the\nadditional issues raised, as detailed below.\n1. Regarding the electrophysiology experiments:\na. While it’s clear that the properly folded Sam50 forms a membrane channel, it is not\nestablished how a channel current and conductance switching would be indicative of “full\nfunctionality” as claimed in the electrophysiology results.\nResponse: As detailed in the response to Reviewer 1, we compared our findings with the\npublished work of Paschen et al., Nature, 2003, where they characterize refolded NcSam50\nusing electrophysiology and voltage-dependent gating, and we showed that we obtained similar\nresults. Our aim here was to confirm whether the POTRA domain was indeed required for\nSam50, i.e., whether full-length Sam50 and POTRA-deleted Sam50 show a similar behavior.\nHence, we have removed “full functionality”, and re-written this section to reflect our exact\nobjective and findings. Additionally, we have included more electrophysiology data in the SI\nFigure S1e-j.\nb. The characterization in the text of the data in fig 1f seems off: the text says both FL and d120\nhave reduction from ~2-3 to ~1 nS at >+-50mV. Figure 1f shows a change from above 3 to\nbelow 3 nS for d120 rather inconsistently with applied voltage, appearing briefly for descending\nvoltage >~200mV, and again for increasing negative voltage from around -400mV to nearly 0.\nThe FL is similar, but with conductance uniformly lower by ~1nS. Additionally, the d120 variant\nappears to have a third conductance state not seen in the FL trace.\nResponse: We believe there is a misunderstanding and we apologize for any confusion. Our\nexperiment and results are as follows:\n The applied voltage is a ramp, and only from +100 mV to –100 mV at 3.33 mV/s. The\napplied voltage is provided on the y-prime axis. This is now colored as red, to match with\nthe graph.\n The y-axis represents the measured current in picoamperes (± 400 pA).\n There are two Sam50 protein channels in the Δ120 experiment, whereas there is only\none protein channel in the full length Sam50, which may have been the source of some\nconfusion. What appears to be “a third conductance state” is, in fact, the response of the\nsecond protein to the applied voltage.\n Sam50 is not a conventional ion channel, unlike VDACs which are also in the outer\nmitochondrial membrane. The “gating-like” behavior of this protein is poorer than VDAC\nand less consistent, and hence, the observed differences.\nWe have now clearly specified the number of protein channels in the graph itself.\nc. Overall, these data seem not to contribute much to the rest of the (very detailed) study of the\nfolding and structure of Sam50.\nResponse page: 5\nResponse: We understand the reviewer’s perspective. Our aim here was to compare the\nbiochemical, spectroscopic, electrophysiology, and in vivo behavior of Sam50 with and without\nits POTRA domain. We show that POTRA may be dispensable for Sam50, and then proceed to\ncharacterize the folding and structure of the POTRA-less Sam50. Hence, we have presented a\ncomparison of results from both constructs in Figure 1. The rephrased sentences in the first\nsection of the results are as follows:\n1st paragraph (page 4, lines 5-9):\n“First, we asked if the TMD was sufficient for Sam50 folding, stability, and\nfunction. To address this, we generated a POTRA-less variant of yeast Sam50\n(Sam50Δ120, only TMD; referred to hereafter as Sam50) (Figure 1a). We\nexamined if the structural and functional features of the full-length Sam50\n(Sam50FL) were retained in POTRA-less Sam50.”\n2nd paragraph last sentence (page 4, lines 26-27):\n“Taken together, our results show that the TMD is sufficient for Sam50 folding\nand function, both in vitro and in vivo.”\n2. Regarding the molecular dynamics simulations:\na. The first paragraph of the section “β-Barrel stability allosterically regulates protein dynamics”\nconcludes that “POTRA can regulate gate dynamics, and may drive the β1-β3 switch…” This\nmight match the more classical definition of allostery, but it’s difficult to understand how β-barrel\nstability regulating gate dynamics would be considered allostery.\nResponse: We agree. We only wanted to point out that strand tilt at β1-β3 changes depending\non whether POTRA is present. We have now rephrased it as follows (page 5, lines 37-39):\n“Therefore, POTRA may help allosterically modulate the β1-β3 switch between\ngate-open and gate-closed conformations.”\nb. Our prior comment on error shading in fig 3c seems to have been misinterpreted; it should be\npossible to show the error on the mean for the RMSF curves in 3c, e.g. by bootstrapping, so\nthat one can see to what extent the different portions of the curves are different within statistical\nerror.\nResponse: We apologize for the misinterpretation of the previous comment. We have presented\nthe RMSF curves with errors in the supporting information (Figure S13a).\nc. Caption of 3b says that FL to d120 results in a decrease in T3 “dynamicity” by >1nm. I would\nthink dynamicity would relate to changes or fluctuations. Neither of those two traces show much\ndynamicity, the change from FL to d120 just shows a change in a rather static distance.\nResponse: We agree. We have now rephrased it as follows (page 22, lines 6-8):\n“(b) Distance analysis between Cα of midplane W240 (β7) and P228 (T3) (marked\non the structure). POTRA deletion (black (FL) versus blue (Δ120)) causes a static\nloss of >1 nm.”\nd. Fig 3c: The emphasis on turn 3 seems to show that while FL and d120 do not fluctuate much\nhere, the two mutants do, which agrees with the result of 3b. Loop 4 seems to be shown as a\nResponse page: 6\ncounter example where no change occurs in mutants. This could be made more clear in the\nfigure/legend.\nResponse: We welcome this suggestion. This is now added in the legend, as follows (page 22,\nlines 13-15):\n“Note how L4 dynamicity is not significantly altered by the mutation, while T3\ndynamicity is high in WF and SNNP. The latter is also reflected in the distance\nanalysis in (b).”\ne. Fig 3e is still not informative. How do these FEL connect to the point about conformational\ngate opening/closing. They report ‘a broader FEL’ in WF-d120. Despite a FEL directly reporting\non the relative probability of sampled configurations, they are not used in this way. The color\nbars also do not seem to agree with the labels on the z-axis, where d120 reaches 9kJ/mol by\neye, but is colored orange. If the FEL is not a main point, as the authors acknowledged in their\nresponse, then why are they being shown in the main text?\nResponse: We understand the reviewer’s perspective. We have now removed the FEL profiles\nfrom Figure 3 and limited the discussion in the text.\n3. In the author's response, they state “Yes, by and large, free energies are equilibrium\nmeasurements. However, for membrane proteins, it has been demonstrated through several\nstudies that kinetic components also contribute to the measured free energy”. This suggests a\nmisunderstanding of what free energy is. We are not arguing that kinetic trapping cannot occur\nin protein folding, nor that this can impact how measurements report on the probability of\nsampled states. However, do not refer to it as a free energy when that is defined only for an\nequilibrium ensemble. If kinetic (time-dependent) elements impact the measured distribution of\nsampled states, then one is not measuring a free energy. This is non-negotiable. When the\nauthors abuse precise, thermodynamic terminology, it undermines the study. Free energies are\nby definition controlled entirely by the Boltzmann distribution, and not by any time-dependent\nfactors. The reworded statement does now avoid making this assertion. However, one could\nargue that there is no need to use the word ‘kinetic stability’,\nwhen kinetic or time-dependent on its own will suffice, although this is not an essential change.\nIf hysteresis occurs, this cannot be an equilibrium transition between states, as equilibrium\ntransitions are not dependent on paths/history. Or at least the measurements are not capable of\nsampling the equilibrium transitions.\nResponse: We apologize for not understanding the reviewer’s earlier concern. We fully agree\nthat when time-dependent factors are in play, the free energy measured is no longer under true\nequilibrium. We have now made the following corrections:\n Equilibrium Gibbs free energy Δ𝐺0 is now replaced with the apparent change in folding\neq\nfree energy Δ𝐺0 at 0 M denaturant.\nF\n The change in Gibbs free energy upon mutation (ΔΔ𝐺0) is now indicated as an apparent\nF\nparameter.\n The phrase “kinetic stability” has been replaced throughout with “kinetic contribution”.\nThese changes are also made considering the terminologies previous publications –reporting\nthe thermodynamics and kinetics of bacterial OMPs– have used. Examples: Radford group\n(Hyusmans et al., PNAS, 2010), Fleming group (Moon et al., JMB, 2011 and PNAS, 2013).\nResponse page: 7\nThese bacterial OMPs achieve “equilibrium” over several hours (12-40 h) and kinetic\ncontributions are discussed. Furthermore, the Kleinschmidt group (Pocanschi et al., Biophys J,\n2006), considers it as an apparent process, as it takes several days to reach reversible\nfolding/unfolding. The nature of membrane mimetics used (micelles versus LUVs, lipid chain\nlength, membrane composition, etc.) also play a role in the time the OMP takes to attain\nequilibrium. Hence, we have now additionally cited these articles in our revision. We have also\nmentioned explicitly that the complex interplay between the protein and the membrane (LUVs)\nprecludes a simple interpretation of the parameters we measure for Sam50. We hope that these\nchanges now adequately address the reviewer’s concerns.\na. In the SI, it is remarked that the kinetic free energy is “deduced” from fits to SI equation 6,\nwhich does not contain that variable on its own. Is it defined from the ratio kU/kF being taken\nequal to an effective folding dissociation constant?\nResponse: We apologize for the oversight in forgetting to include the equation for calculating the\nchange in kinetic free energy. This is now added as Equation 8 in the SI.\n4. We appreciate that the authors removed the word metastable. Despite this modification the\nauthors insist in their response that “we disagree with the Reviewer’s comment that a\nmetastable state is unfavorable”. It is by its very definition not the free energy minimum, which\nmeans that it is indeed unfavorable relative to the thermodynamic free energy minimum. This is\ntrue whether it is metastable in state space (i.e. a metastable liquid relative to a solid) or\nmetastable within a free energy landscape of an equilibrium ensemble, where a higher energy\nminimum is metastable because of barriers separating it from neighboring states. This minimum\non the FEL can only be termed metastable if it is not the global free energy minimum.\nResponse: We understand the reviewer’s concern, and appreciate the explanation they have\nprovided. We have now re-edited sections of the text that might indicate a “metastable” state or\nstructure for Sam50. We merely state that it appears to be “less stable” than its bacterial\ncounterpart.\n5. Fig 4b and text say that unfolding rates were obtained from interpolation, which seems to be\ntaking the value of the linear fit at a particular concentration of GdnHCl (5.5M and 4.5M). Why\nthose specific concentrations?\nResponse: We chose these values, as they were the approximate midpoints for each arm, and\ntherefore, would help in estimating k and k most accurately. This is now mentioned in the\nu1 u2\nfigure legend and in the SI Methods.\n6. Fig S31 is a helpful illustration of the kinetic model of folding in LUVs that plays a central role\nin the paper; the authors might consider having this as part of figure 4, where the folding and\nunfolding rate measurements are shown. Also regarding Fig S31: There are rates for folding to\nthe transition states, and unfolding from the final structure to the transition states, but not for\nfolding from transition states to final state. Where are the rates that connect the transition state\nto the folded state?\nHow do they know that the unfolding from the folded state returns them to the transition state\nand not to the previous intermediate or the disordered/adsorbed state?\nResponse page: 8\nResponse: We are delighted to note that the reviewer found Figure S31 as helpful, and we have\nnow incorporated it as a part of Figure 4, as suggested.\nRegarding the TS→N rates: We carried out kinetics measurements with the SX20 stopped flow\nset-up (latest model from Applied Photophysics Ltd., UK) equipped with the latest accessories\nthey have. In all the experimental conditions, were able to capture only three folding rates and\ntwo unfolding rates. This suggests that the conversion of the transition state structure to the\nfolded state is either too fast or too slow for the instrument to capture. We are inclined towards\nthe process being too slow, as it also explains the time course measurement data from the\nequilibrium titrations (Figure S5). We are hoping that AFM-based experiments may be able to\nprovide some additional answers in the future.\nRegarding the refolding experiment: Yes, we have carried out both refolding and re-unfolding\nexperiments to assess if the protein returns to the TS. We find that the rates of refolding indeed\nmatch with the folding rates (Figures 3a, S21). Similarly, the re-unfolding and unfolding rates are\ncomparable (Figures 3a, S20). Hence, we are fairly certain that unfolding from the folded state\nreturns to the transition state structures.\n7. Fig 6 caption says G443→A is not tolerated and causes loss in cell viability, but wasn’t it\nestablished earlier that no single point substitutions resulted in loss of cell viability?\nResponse. We apologize for the confusion, as what we stated was incomplete. We meant to\nsay that when present with several other mutations we tested, G443→A emerges as lethal for\nthe cell. We have now added this specific clarification in the figure caption, as follows (page 27,\nlines 8-10):\n“Other residues cannot adopt these unusual backbone torsion angles, explaining\nwhy G443A is not tolerated when present with other mutations, and causes loss\nin cell viability.”\n8. In the referee response, they state “The proposed folding mechanism derived from kinetics\nmeasurements is distinct from the dynamics we obtain for the post-folded structure in our MD\nsimulations, and there is nothing to interrogate in this.” How is there nothing to interrogate when\nthey seem to be stating that the simulations are in disagreement with the experimental\nmeasurements?\nResponse: We believe there may be a misunderstanding here. We only meant to state that we\nare capturing the dynamics of nearly the entire polypeptide chain in the kinetics measurements,\nand that we are capturing the local dynamics (mainly loops, turns etc.) in our MD simulations.\nTherefore, we have now restricted the comparison of our MD simulation results with only the\nresults from equilibrium Δ𝐺0 measurements.\nF\nResponse page: 9\nReviewer #3 (Remarks to the Author):\nI am satisfied with the authors' response to the issues I raised.\nResponse: We thank the reviewer for their time and insights, which have helped improve our\nmanuscript considerably.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and insights. We hope we have now adequately\naddressed all the concerns and clarified the queries raised herein.\nResponse page: 10\nManuscript NCOMMS-25-06253B-Z\nTitle: Linking multipoint β-barrel folding with stability and regulation in the mitochondrial outer\nmembrane protein Sam50\nWe thank the reviewers for taking the time to share their suggestions in detail. We are delighted\nto now submit a thoroughly revised manuscript that incorporates all the suggested changes.\nMoreover, we have included the following experimental data:\n1. Measurements of step-like insertion events for both proteins.\n2. Measurements channel flickering events, with statistics.\n3. Single channel measurements at defined holding voltages, with statistics.\n4. Multichannel measurements, and G/G analysis.\nmax\n5. Other suggested additions in the figures, and replotting wherever needed.\nAdditionally, we have revised the relevant text to explain our findings, and discussed our\nobservations in the context of previous studies (including 10.1016/j.cell.2008.01.028 and other\nworks of Wagner). We are positive that our revisions clearly align with the suggestions you\nprovided, and you will find our revised submission now acceptable for publication in Nature\nCommunications.\nReviewers’ suggestions are in blue font, and Authors' responses highlighting changes made are\nin black font:\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have responded to our comments and suggestions.\nResponse: We thank the reviewer for their time and for their invaluable suggestions. We are\ndelighted to note that the reviewer is satisfied with the changes we had made.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and for their invaluable suggestions.\nReviewer #5 (Remarks to the Author):\nI read the manuscript by Roshika Ravi and coworker entitled with “Multipoint folding and stability\nwith functional regulation in the mitochondrial outer membrane protein Sam50.” Without doubt\nthis is important research area for the community. I also agree with the other reviewers that the\nauthors should strengthen the clarity of their results. As there are only a few characterizations of\nSAM50, the novelty and relevance of your results could be improved (easy to say, I know its\nvery difficult to do so after a lengthy collection of data). In particular your claim (abstract): Our\nfindings suggest how the dynamic structure of Sam50 offers a functional advantage in vivo, …\nResponse page 1\nResponse: We thank the reviewer for taking the time to review our study, appreciating the body\nof work we have done, and for highlighting the significance of our findings. We have now\nmoderated our claim by removing “in vivo” from this sentence and the previous sentence in the\nabstract. We have also addressed the suggestions in our revised submission with additional\nexperiments and statistics.\nI was asked in particular to review the electrophysiology experiments.\nElectrophysiology as shown in this manuscript can reveal membrane channel behavior with\nsingle channel resolution: pore size, flexibility, …. However, single molecules reconstituted in an\nartificial membrane system lack the biological environment and have poor statistics. In particular\nthe question of the right lipid composition is often raised but mostly this is not a very crucial\nparameter. Diphytanoyl-PC as used here is typically used and has no phase transition in the\nrange of investigation.\nResponse: We have used diphytanoyl-PC, which has been used in all previous\nelectrophysiology measurements of Sam50/Tob55 and other mitochondrial OMPs. However, we\nagree that single molecule measurements have poor statistics. To circumvent this, we captured\nmultichannel gating characteristics as well, at different voltages. The data with statistics are\nprovided in Figure S1p, and display typical bell-shaped profiles for both Sam50 FL and Δ120.\nFig 1f: Here the authors compare the full SAM50 with the truncated one. Clearly both types\n(FL/delta) insert into lipid membranes showing related channel conductance. However,\ninasmuch the conductance level is a proof for the functionality has not been discussed. It’s a bit\nunfortunate to compare an experiment with two (or more) inserted channels (delta) with one (or\nmore) channel (FL). Also, voltage ramps can hide slower kinetics but here it seems to be ok (but\nmaybe confusing for outside readers. I see for the black line one fully open channel and one\nwhich opens-closes, the blue one I could interpret as three (or more) states. Careful inspection\nof the conductance shows either a difference in conductance between both types of porins\n(FL/delta) or a broader distribution. The truncated one show also much more flickering. The data\nin the supplement S1 could be used to get more insight.\nResponse: We welcome the suggestions. We have now incorporated the following additions:\n1. We now compare one channel of FL with one channel of Δ120 in the voltage ramp\nmeasurement. The data is in Figure 1g.\n2. We additionally provide holding voltage measurements to obtain clear data on the\nchannel flickering (Figure 1f; Figure S1i-j). We have labeled the two conductance states\nas O1 and O2, as done by Wagner–Pfanner–Wiedemann in 10.1016/j.cell.2008.01.028.\n3. We have calculated the sampling frequency of the two conductance states O1 and O2,\nfor FL and Δ120 at a specific holding voltage, to compare flickering (Figure 1f; Figures\nS1i-j).\n4. We now discuss conductance levels with channel dynamicity and functionality on page 4\n(lines 19-25) of the Results, and page 11 (lines 12-28) of the Discussion, in the revised\nmanuscript.\nMy conclusion would be that the truncated version is more flexible compared to the full length.\nOn the other hand, a clear conclusion requires a larger number of experiments. Likely the\nResponse page 2\nauthors have many more results, so, maybe adding a sentence to clarify the observation. Also,\nprevious work (ref 11 and other works by R. Wagner) showed also flickering channel. Maybe\nthis should be discussed in more depth.\nResponse: We agree that the truncated Sam50 (Δ120) may be more flexible than FL. POTRA\nmay also contribute to deciding this flexibility – indeed, our MD simulations provide evidence for\nPOTRA’s contribution to the β-barrel dynamicity (see Figures 3a).\nAs suggested by the reviewer, we have now included additional results, and carried out\ncomparisons and statistics for the flickering in both channels (Figures S1e-j). In addition, we\nhave now compared our observations with Wagner’s works in the revised manuscript on page 4\n(lines 19-25) of the Results, and page 11 (lines 12-28) of the Discussion, in the revised\nmanuscript.\nFor my own taste and to avoid confusion for the general reader I would add the control (green)\nto the supplement. Clearly such a control is needed, you never start a reconstitution without\nhaving a nearly zero conductance corresponding of a tight membrane.\nResponse: We agree, and you can rest assured that we always carry out experiments only after\nwe obtain a tight membrane with a near-zero conductance. As suggested, we have included the\ncontrol (green) in Figures S1e-f. We have also included the control in Figure 1g.\nOverall, the electrophysiological experiments are a good control on possible functional\nreconstitution. The data could be more in depth evaluated and compared to previous findings\n(ref 11 and R. Wagner publishing with the same team).\nResponse: We welcome the suggestion. We now provide more analyses corresponding to\ngating transitions and associated conductance, and flicker frequency. Additionally, we discuss\nand compare our findings with the previous reports (particularly the Wagner–Wiedemann–\nPfanner team).\nResponse page 3"}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have responded to our comments and suggestions.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I read the manuscript by Roshika Ravi and coworker entitled with “Multipoint folding and stability with functional regulation in the mitochondrial outer membrane protein Sam50.” Without doubt this is important research area for the community. I also agree with the other reviewers that the authors should strengthen the clarity of their results. As there are only a few characterizations of SAM50, the novelty and relevance of your results could be improved (easy to say, I know its very difficult to do so after a lengthy collection of data). In particular your claim (abstract): Our findings suggest how the dynamic structure of Sam50 offers a functional advantage in vivo, …\nI was asked in particular to review the electrophysiology experiments.\nElectrophysiology as shown in this manuscript can reveal membrane channel behavior with single channel resolution: pore size, flexibility, …. However, single molecules reconstituted in an artificial membrane system lack the biological environment and have poor statistics. In particular the question of the right lipid composition is often raised but mostly this is not a very crucial parameter. Diphytanoyl-PC as used here is typically used and has no phase transition in the range of investigation.\nFig 1f: Here the authors compare the full SAM50 with the truncated one. Clearly both types (FL/delta) insert into lipid membranes showing related channel conductance. However, inasmuch the conductance level is a proof for the functionality has not beed discussed. It’s a bit unfortunate to compare an experiment with two (or more) inserted channels (delta) with one (or more) channel (FL). Also, voltage ramps can hide slower kinetics but here it seems to be ok (but maybe confusing for outside readers. I see for the black line one fully open channel and one which opens-closes, the blue one I could interpret as three (or more) states. Careful inspection of the conductance shows either a difference in conductance between both types of porins (FL/delta) or a broader distribution. The truncated one show also much more flickering. The data in the supplement S1 could be used to get more insight.\nMy conclusion would be that the truncated version is more flexible compared to the full length. On the other hand, a clear conclusion requires a larger number of experiments. Likely the authors have many more results, so, maybe adding a sentence to clarify the observation. Also, previous work (ref 11 and other works by R. Wagner) showed also flickering channel. Maybe this should be discussed in more depth.\nFor my own taste and to avoid confusion for the general reader I would add the control (green) to the supplement. Clearly such a control is needed, you never start a reconstitution without having a nearly zero conductance corresponding of a tight membrane.\nOverall, the electrophysiological experiments are a good control on possible functional reconstitution. The data could be more in depth evaluated and compared to previous findings (ref 11 and R. Wagner publishing with the same team)."}, "author_response": "We thank the reviewers for taking the time to share their suggestions in detail. We are delighted\nto now submit a thoroughly revised manuscript that incorporates all the suggested changes.\nMoreover, we have included the following experimental data:\n1. Measurements of step-like insertion events for both proteins.\n2. Measurements channel flickering events, with statistics.\n3. Single channel measurements at defined holding voltages, with statistics.\n4. Multichannel measurements, and G/G analysis.\nmax\n5. Other suggested additions in the figures, and replotting wherever needed.\nAdditionally, we have revised the relevant text to explain our findings, and discussed our\nobservations in the context of previous studies (including 10.1016/j.cell.2008.01.028 and other\nworks of Wagner). We are positive that our revisions clearly align with the suggestions you\nprovided, and you will find our revised submission now acceptable for publication in Nature\nCommunications.\nReviewers’ suggestions are in blue font, and Authors' responses highlighting changes made are\nin black font:\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have responded to our comments and suggestions.\nResponse: We thank the reviewer for their time and for their invaluable suggestions. We are\ndelighted to note that the reviewer is satisfied with the changes we had made.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and for their invaluable suggestions.\nReviewer #5 (Remarks to the Author):\nI read the manuscript by Roshika Ravi and coworker entitled with “Multipoint folding and stability\nwith functional regulation in the mitochondrial outer membrane protein Sam50.” Without doubt\nthis is important research area for the community. I also agree with the other reviewers that the\nauthors should strengthen the clarity of their results. As there are only a few characterizations of\nSAM50, the novelty and relevance of your results could be improved (easy to say, I know its\nvery difficult to do so after a lengthy collection of data). In particular your claim (abstract): Our\nfindings suggest how the dynamic structure of Sam50 offers a functional advantage in vivo, …\nResponse page 1\nResponse: We thank the reviewer for taking the time to review our study, appreciating the body\nof work we have done, and for highlighting the significance of our findings. We have now\nmoderated our claim by removing “in vivo” from this sentence and the previous sentence in the\nabstract. We have also addressed the suggestions in our revised submission with additional\nexperiments and statistics.\nI was asked in particular to review the electrophysiology experiments.\nElectrophysiology as shown in this manuscript can reveal membrane channel behavior with\nsingle channel resolution: pore size, flexibility, …. However, single molecules reconstituted in an\nartificial membrane system lack the biological environment and have poor statistics. In particular\nthe question of the right lipid composition is often raised but mostly this is not a very crucial\nparameter. Diphytanoyl-PC as used here is typically used and has no phase transition in the\nrange of investigation.\nResponse: We have used diphytanoyl-PC, which has been used in all previous\nelectrophysiology measurements of Sam50/Tob55 and other mitochondrial OMPs. However, we\nagree that single molecule measurements have poor statistics. To circumvent this, we captured\nmultichannel gating characteristics as well, at different voltages. The data with statistics are\nprovided in Figure S1p, and display typical bell-shaped profiles for both Sam50 FL and Δ120.\nFig 1f: Here the authors compare the full SAM50 with the truncated one. Clearly both types\n(FL/delta) insert into lipid membranes showing related channel conductance. However,\ninasmuch the conductance level is a proof for the functionality has not been discussed. It’s a bit\nunfortunate to compare an experiment with two (or more) inserted channels (delta) with one (or\nmore) channel (FL). Also, voltage ramps can hide slower kinetics but here it seems to be ok (but\nmaybe confusing for outside readers. I see for the black line one fully open channel and one\nwhich opens-closes, the blue one I could interpret as three (or more) states. Careful inspection\nof the conductance shows either a difference in conductance between both types of porins\n(FL/delta) or a broader distribution. The truncated one show also much more flickering. The data\nin the supplement S1 could be used to get more insight.\nResponse: We welcome the suggestions. We have now incorporated the following additions:\n1. We now compare one channel of FL with one channel of Δ120 in the voltage ramp\nmeasurement. The data is in Figure 1g.\n2. We additionally provide holding voltage measurements to obtain clear data on the\nchannel flickering (Figure 1f; Figure S1i-j). We have labeled the two conductance states\nas O1 and O2, as done by Wagner–Pfanner–Wiedemann in 10.1016/j.cell.2008.01.028.\n3. We have calculated the sampling frequency of the two conductance states O1 and O2,\nfor FL and Δ120 at a specific holding voltage, to compare flickering (Figure 1f; Figures\nS1i-j).\n4. We now discuss conductance levels with channel dynamicity and functionality on page 4\n(lines 19-25) of the Results, and page 11 (lines 12-28) of the Discussion, in the revised\nmanuscript.\nMy conclusion would be that the truncated version is more flexible compared to the full length.\nOn the other hand, a clear conclusion requires a larger number of experiments. Likely the\nResponse page 2\nauthors have many more results, so, maybe adding a sentence to clarify the observation. Also,\nprevious work (ref 11 and other works by R. Wagner) showed also flickering channel. Maybe\nthis should be discussed in more depth.\nResponse: We agree that the truncated Sam50 (Δ120) may be more flexible than FL. POTRA\nmay also contribute to deciding this flexibility – indeed, our MD simulations provide evidence for\nPOTRA’s contribution to the β-barrel dynamicity (see Figures 3a).\nAs suggested by the reviewer, we have now included additional results, and carried out\ncomparisons and statistics for the flickering in both channels (Figures S1e-j). In addition, we\nhave now compared our observations with Wagner’s works in the revised manuscript on page 4\n(lines 19-25) of the Results, and page 11 (lines 12-28) of the Discussion, in the revised\nmanuscript.\nFor my own taste and to avoid confusion for the general reader I would add the control (green)\nto the supplement. Clearly such a control is needed, you never start a reconstitution without\nhaving a nearly zero conductance corresponding of a tight membrane.\nResponse: We agree, and you can rest assured that we always carry out experiments only after\nwe obtain a tight membrane with a near-zero conductance. As suggested, we have included the\ncontrol (green) in Figures S1e-f. We have also included the control in Figure 1g.\nOverall, the electrophysiological experiments are a good control on possible functional\nreconstitution. The data could be more in depth evaluated and compared to previous findings\n(ref 11 and R. Wagner publishing with the same team).\nResponse: We welcome the suggestion. We now provide more analyses corresponding to\ngating transitions and associated conductance, and flicker frequency. Additionally, we discuss\nand compare our findings with the previous reports (particularly the Wagner–Wiedemann–\nPfanner team).\nResponse page 3"}]

Decision letter