biomolecules
Review

Functional Implications of Dynamic Structures of Intrinsically
Disordered Proteins Revealed by High-Speed AFM Imaging
Toshio Ando
Nano Life Science Institute, Kanazawa University, Kanazawa 920-1192, Japan; tando@staff.kanazawa-u.ac.jp

Abstract: The unique functions of intrinsically disordered proteins (IDPs) depend on their dynamic
protean structure that often eludes analysis. High-speed atomic force microscopy (HS-AFM) canconduct this difficult analysis by directly visualizing individual IDP molecules in dynamic motion
at sub-molecular resolution. After brief descriptions of the microscopy technique, this review firstshows that the intermittent tip–sample contact does not alter the dynamic structure of IDPs and thendescribes how the number of amino acids contained in a fully disordered region can be estimated fromits HS-AFM images. Next, the functional relevance of a dumbbell-like structure that has often been
observed on IDPs is discussed. Finally, the dynamic structural information of two measles virus IDPs
acquired from their HS-AFM and NMR analyses is described together with its functional implications.
Keywords: intrinsically disordered proteins; IDPs; intrinsically disordered regions; IDRs; high-speed
AFM; dynamic structure; bioimaging

Citation: Ando, T. Functional
Implications of Dynamic Structuresof Intrinsically Disordered Proteins
Revealed by High-Speed AFM
Imaging. Biomolecules 2022, 12, 1876.
https://doi.org/10.3390/
biom12121876
Academic Editors: Elisar Barbar,
Nathalie Sibille and Vladimir
N. Uversky
Received: 28 November 2022
Accepted: 11 December 2022
Published: 14 December 2022
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with regard to jurisdictional claims inpublished maps and institutional affiliations.

Copyright:© 2022 by the author.

Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
In physiological conditions, intrinsically disordered proteins (IDPs) are devoid of awell-defined three-dimensional structure entirely or partly. And yet, they can carry out
biological functions and often work in the regulation of transcription and translation andin cellular signal transduction [1,2]. Therefore, IDPs are more abundant in eukaryotic cells
than in prokaryotic cells. Interestingly, IDPs are relatively abundant in viruses [3,4]; Sonia
Longhi and colleagues first found a viral IDP in the measles virus [5]. This abundance
relative to the total number of viral proteins is possibly because viruses have highjacked
the cellular machinery of their hosts. The intrinsically disordered regions (IDRs) containedin IDPs are highly protean. The residues in an IDR responsible for weak intramolecularinteractions are widely distributed in space and time. Therefore, IDRs can dynamicallysample multiple conformations, including fully disordered tail-like structures, loosely
folded conformations, and incompletely or fully folded secondary structures. The residues
responsible for target binding are also distributed in space and time, enabling an IDP
to bind multiple targets by structural adaptation, resulting in different folded structuresby coupled folding, as exemplified in the binding of an IDP (p53) to different structured
proteins [6]. Therefore, IDPs can be multifunctional. The coupled folding of an IDR
induced by binding to structured proteins [7] may imply that strong binding requires
folding of the IDR. However, dimerization or target binding of IDRs without folding has
been observed [8,9]. Their dissociation constants (Kd ) are in the order of 10 µM. A recent
study has suggested that two highly disordered IDPs can bind each other with much higher
affinity while retaining their structural disorder and highly dynamic character [10,11].
Moreover, the flexible and adaptable nature of IDRs bestows on them high sensitivity toenvironmental changes and post-translational modifications. That is, the structure of IDPs
and their inter-molecular interactions can be altered by minute changes in the intracellularenvironment and post-translational modifications. The biological importance of such highsensitivity is typically exemplified by the formation of liquid droplet-like condensates(membrane-less organelles) by liquid-liquid phase separation (LLPS) [12–16]. Thus, theBiomolecules 2022, 12, 1876. https://doi.org/10.3390/biom12121876

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flexible, dynamic structural nature of IDRs is the basis for IDPs’ distinct functions andfunctional mechanisms that cannot be achieved by structured proteins.
As such, elucidating the conformational ensemble and dynamics of IDPs is essential
for understanding their functional mechanisms. However, various techniques developed
and used for studying the structure of structured proteins are often not instrumental
in the structural analysis of IDPs. The two major structural biological techniques, Xray crystallography and cryogenic electron microscopy (cryo-EM), do not have practicalapplications for IDPs because IDRs hamper crystallization and are too thin to be visualized
by cryo-EM. Two ensemble-averaging methods, small-angle X-ray scattering (SAXS) [17,18]
and solution NMR spectroscopy [19], have mainly been used for structural analysis of IDPs
(for their approximate performances, see Table 1). Various SAXS-derived data, such as
radius of gyration (Rg) and Kratky plot, provide information about the overall structuralproperties of IDPs. However, distinguishing individual structures of an IDP coexistingin solution and quantifying their populations require modeling of structural ensembles.
For modeling, several methods have been developed, such as the ensemble optimization
method (EOM) [20,21], ILBOMD [22], and MultiFoXS [23]. However, the resolution of
SAXS is low (usually ~5 nm) [18,24], and the unique determination of multiple structures
and their populations from SAXS data alone is difficult. Therefore, SAXS analysis is usuallycombined with molecular dynamics simulations or NMR analysis. In contrast to SAXS,NMR cannot determine the overall structure of IDPs. Instead, NMR determines highresolution local structures of folded regions, transiently appearing structured regions, andresidues interacting with targets using two-dimensional 1 H-15 N/1 H-13 C heteronuclear
single quantum correlation spectra. This also means that NMR can specify disorderedregions in an IDP that do not interact with other residues. Importantly, NMR allows modelbased population analysis for multiple conformational states. A recent study proposed an
improved NMR method for ensemble analysis based on solvent paramagnetic relaxation
enhancement, enabling accurate, high-sensitive detection of low populations of residual
structure [25]. However, NMR is still poor at distinguishing fully disordered and loosely
folded structures. Loosely folded structures are often identified wrongly as fully disordered.
Besides, there is an upper limit of analyzable molecular weight (~40 kDa). For larger IDPs,
their deletion constructs with smaller molecular weights have to be subjected to NMR
analysis. However, the analysis in this way does not necessarily guarantee that the revealed
structures of segments are identical to those in the full-length protein. This is because
intramolecular interactions within an IDP facilitated by a flexible IDR can alter the IDP
structure, as has been demonstrated for Atg13 [26,27]. Moreover, it is hard to determine
the rates of transitions between different conformational states that occur within >~100 ms,
although the fast motion of individual residues can be detected by NMR relaxation analysis
without information on structural coordinates [28].
Table 1. Comparison of four techniques used in structural analysis of proteins.
Data Collection
Time

Sample AmountNMR (a)SAXS

days–months–h (c)2–100 mg
0.1–10 mg

HS-AFM~10 s

0.1 µgAFM

min

0.1 µgSpatialResolution~0.2 nm
1–10 nm (d)2–3 nm (XY)
0.1 nm (Z)
2–3 nm (XY)
0.1 nm (Z)Dynamics

Molecular Weight
limitation

ps–ms (b)s-min

<40 kDanone

20–100 ms (e)

none

>30 s

none

(a) Solution NMR spectroscopy; (b) Obtainable dynamics do not necessarily include structural coordinates;(c) Largely depends on the power of X-ray source (long exposure to X-ray damages proteins); (d) Usually ~5 nm.

Revealed molecular shapes are model dependent; (e) 20–100 ms for IDPs and ~100 ms for structured proteins.

As a complement to the limited capability of these ensemble-averaging methods,
we need a single-molecule method that can observe the dynamic protean structure of

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individual IDP molecules, even at a spatial resolution lower than NMR. Conventional
atomic force microscopy (AFM) was previously used to characterize IDPs. However, its
target was aggregated assemblies of an IDP or constructs of structured domains within
IDPs, such as the nonstructural protein 3 (a protease with folded structure, unlike thename of this protein) from the hepatitis C virus [29] and the DNA binding domain of
p53 [30]. Crystal structures of these domains have been obtained. Single molecules of
IDPs with disordered regions of >20 residues have never been successfully imaged withconventional AFM. Therefore, we have explored the potential of high-speed AFM (HSAFM) in the dynamic structural analysis of IDPs. This relatively young microscopy has
been successfully used to visualize the dynamic behavior of structured proteins during
their functional activity at 2–3 nm lateral and ~0.1 nm vertical spatial resolution and at~100 ms temporal resolution under near-physiological conditions, without any chemicallabeling (Table 1), as demonstrated for a variety of structured proteins [31–35]. IDPs
have also been successfully visualized by HS-AFM: e.g., facilitates chromatin transcription(FACT) protein [36,37], the archaeal Hef protein [38], and the bacterial flagellar hook-length
control protein FliK [39]. These studies have demonstrated the capability of HS-AFM tovisualize the thin structure of IDRs in dynamic motion. Importantly, as demonstrated ina recent study [40], HS-AFM can determine approximately the number of amino acids
contained in a fully disordered IDR. Thus, HS-AFM can delineate and quantify the dynamic
structure of IDPs, as demonstrated for several IDPs, including viral proteins [40]. Moreover,function-related phenomena of IDPs have also been visualized in recent HS-AFM studies:e.g., ion-dependent structural transitions of a stator ring of the bacterial flagellar motor
between unfolded and folded states [41], amyloid fibril formation from α-synuclein [42]and Sup35 [43], the assembly process of translation factors around the ribosomal stalkcomplex [44] and the formation of the droplet-like pre-autophagosomal structure (PAS) via
LLPS [14]. In this review, some recent HS-AFM studies on IDPs, including two measles
virus IDPs and those with a dumbbell-like shape, are described after a brief introduction of
HS-AFM and verification of the absence of AFM-tip disturbance to IDP structure.
2. HS-AFM Imaging in Amplitude Modulation ModeIn the amplitude modulation mode, the AFM cantilever oscillates in the Z-direction
so that the sharp tip attached to the free end of the cantilever contacts with the sample
surface intermittently. This intermittent contact can avoid sample dragging during lateralscanning of the sample stage relative to the tip. Upon tip-sample contact, the cantilever
oscillation amplitude is reduced. To maintain the contact (tapping) force constant, feedback
control moves the sample stage in the Z-direction so that the oscillation amplitude returns
to its set point value. As a result, the motion of the sample stage traces the sample surface(the Z-motion direction is opposite to the sample height). Therefore, we can construct the
sample surface topography using a computer from the signal that drives the sample stage
in the Z-direction. In our HS-AFM system, all instrumental components, including thecantilever, the scanner, and all electronics, are optimized for highly frequent tip-sample
contact, fast conversion of the cantilever deflection signal to the amplitude signal, fast
data sampling, fast scanning of the sample stage, or fast feedback control. To minimize
the tip-sample contact strength, the free oscillation amplitude A0 of a short cantilever(7–10 µm) with a spring constant of 0.1–0.2 N/m is set at 1–2 nm, and its set point is set at
0.85–0.9 × A0 , resulting in the cantilever’s oscillation energy loss per tap is only a few kB T
on average (kB , Boltzmann constant; T, the temperature in kelvin). The feedback control
speed depends on the ratio of A0 to the sample height hs ; i.e., the control speed increases
with increasing A0 /hs . Since the height of IDPs is generally small (the mean height of afully disordered IDR is about 0.5 nm), the imaging speed can be increased up to 50 fps
for many IDPs. Very recent improvements to our HS-AFM system have enabled ~70 fps
even for structured proteins with hs < 25 nm, although this improved system has not yet
been used for IDPs. For HS-AFM imaging of biological molecules, they have to be placedon a substrate surface (Figure 1a). HS-AFM imaging of IDPs requires the surface to beBiomolecules 2022, 12, 1876

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Biomolecules 2022, 12, x FOR PEER REVIEW
5 of 18hydrophilic and flat at the atomic level. Practically, bare mica that meets these requirements

is probably the only choice. In general, IDRs only weakly attach to mica via electrostaticand van der Waals interactions and, therefore, exhibit fast Brownian motion on mica. Whenan
IDP is independency
disordered overentire length,distribution
it diffuses too
fast tothat
be imaged
even
at 50
fps.
intervalof the displacement
suggests
the shapeof theIDR
Inchanges
such a muchcase, fasterwe needfuse
a foldedprotein(e.g.,
to the N- function
or C-terminus
thantothe
imageacquisition
rate.
The GFP)
autocorrelationof R2D(t)oftheIDP
as except
an anchor
onto
More thedetailsof practicalin HS-AFM
imagingwaszero
for Δt= 0,mica.
even when
HS-AFM
imagesissues
were captured
at 50
fps [40].of
While
the
tip
intermittently
contacts
thesample,themica
surfaceisalwaysincontact
with
IDPs are described in [45]. For more comprehensive descriptions of the fundamentals,
the sample. andTherefore,
the mica
surface may
have a non-negligible
effect, especiallyon thetechniques,
biologicalapplicationsof HS-AFM,
a book is available[46]. HS-AFM
highly flexible
structureof commercialized
IDRs. An IDR is confineda 2D solution(RIBM,
space onthe mica
systemshave alreadybeen
by three tomanufacturers
Bruker-JPK,
surface,entailing(R2D) longer thanShortthe meanthree-dimensional(3D)
end-to-end
distance
andOxfordInstruments-Asylum).
cantilevers
with a spring
constantof 0.1–0.2
N/m
in solution,(R3Dfrequency). However,similarconfinement
canwateralso occur
in thecrowded cytoplasm,
anda resonant
higherthan
400 kHz in
are alsocommerciallyavailable
nucleus,and viralandcapsid.
from
NanoWorld
RIBM.

Figure1.1.Absence
Absenceofoftip
tipdisturbance
disturbancetotothe
thestructure
structureofofIDPs.
IDPs.(a)(a)Schematic
Schematicshowing
showingHS-AFM
HS-AFMimaging
imagFigure
ing of IDP molecules placed on mica surface. (b–d) Molecular features of PQBP-1 (1–214) measuredof IDP molecules placed on mica surface. (b–d) Molecular features of PQBP-1 (1–214) measured
by HS-AFM at various imaging rates (6.7–50 fps): (b) Mean height of N-terminal globule, (c) mean
byheight
HS-AFM
at various
rates(6.7–50 fps):(b) Meanheightof N-terminal(c) meanof IDR,and (d)imaging
mean 2D
end-to-end
distanceof IDR.
(e) Distributions
ofglobule,C-terminal
endheight
of
IDR,
and
(d)
mean
2D
end-to-end
distance
of
IDR.
(e)
DistributionsofC-terminal
end
displacements in the X-direction were observed for WT PQBP-1 during time periods of Δt and 5Δt.
displacementsintheX-direction
wereobservedfor
WT
PQBP-1duringtime
periods
of
∆t
and
5∆t.
Δt is one frame acquisition time (42 ms). The numbers shown on each panel are the mean displace-∆tin theacquisition+X and −Xtime
directions.
the IDR
withinis constantly
disordered.
isments
one frame(42 ms).Note
Thethatnumbers
shown
onPQBP-1
each panel
are the mean
displacementsin the +X and −X directions. Note that the IDR within PQBP-1 is constantly disordered.

4. Constancy of Flexibility/Rigidity of Fully Disordered Regions
3. No Except
Effect ofContact ondenatured
the Structureof IDPs chains are known to haveforTip-Sample
rare cases, chemicallypolypeptide

The flexibility
tip-sampleorinteraction
is much weakerinaminoliquidsacidthansequencein air becauseof the lack of
uniquerigidity, irrespectiveof theand composition
capillarity
in liquids[47].
Nevertheless,there still
be arelationshipconcern thatthe physical[48]. Thisforces
constancy
can beexpressed
by a powerlaw may
for thebetweenthecontact
a protein
and anoscillating
tip in 〈Rang〉:aqueous
numberbetweenof aminoacids Naamoleculeand the mean
radiusof gyration,i.e., 〈Rg〉solution= 0.1927 would
nm ×
have
a strong
impactonIn
thestructureand functionof the formolecule.
The absence
ofchains,
such anNaa0.598(Figure2a) [48].
the
3D worm-likechain modelsemi-flexible
polymer2
impacthas
beenverifiedforvariousstructured
proteins.
For
example,the
HS-AFM
imagesthe mean square of Rg, i.e., 〈Rg 〉, can be expressed as a function of the 3D persistence lengththat
capturedtheprocessive
movementof
myosinVonactin
filaments
at7
fps
showedP3D asthat its movement velocity is identical to3that measured
by fluorescence microscopy in the
4
𝑃3𝐷 𝐿
2𝑃
2𝑃3𝐷
𝐿
2 The 3𝐷
same buffer solutionchangesof〈𝑅𝑔2condition〉=‒[31].
𝑃3𝐷+ conformational‒ 2 × [1‒ Exp
(‒
)] F
, (1)
1 -ATPase filmed with
3 40 s were𝐿observed
𝐿
3𝐷
HS-AFM at 12.5 fps for overto continue𝑃without
the changes being
where L is[32].
the contourof a polymer
chain. In werethe caseof polypeptide
chains,
L canofdebilitatedDuring length
this imaging,the molecules
tappedwith the AFM
tip tens
millionsof times.
However, theabsencea tip
disturbanceto thedynamic
structure
be calculated
approximately
as L= 0.360ofnm
× (Naa–1); 0.360 nm
is theaveragedistanceof
IDPs
cannotbeadjacentverified amino
straightforwardly
becausedynamic
conformationalchanges
betweentwo
acids [49,50].
The 〈Rtheg〉 dataof chemicallydenaturedpoly-ofIDPs
occur
thermallyandstochastically.
peptide chains approximately followed Eq. (1), suggesting that chemically denatured protheworm-likefeedback control
error thisis generally
small
foroflow
samplestowith
teinsAlthoughbehave likechains. Fromfitting, thevalue
P3D heightis determinedbehs0.976≤ A0nm.
, the error becomes larger (and hence the tip-sample interaction force becomesstrongerin both Xand Z-directions)withincreasing X-scanningvelocity. 〈RTherefore,the
In contrast
to chemicallydenaturedpolypeptides,
the SAXS-measuredg〉 values of
best
way toexaminethe possible
tip disturbanceis to comparethecharacteristic
72 IDRs
without
permanent
secondarystructuresto
inIDPsthe native
state havebeenreported
structuralparameters
an IDP
fromdetailed
its images
filmedanalyses
at various
not to follow
a uniqueof
power
lawquantified[51]. However,structuralon scanningsome ofvelocities.
Polyglutamine
tract-binding
protein
1(PQBP-1)anditsdeletionconstruct
PQBPthese IDRs have identified regions that tend to form residual structures [52–55]. In contrast
1 to(1–214)
were
subjected
tothis
examination[40].
PQBP-1has
anN-terminal
globular
SAXS, HS-AFM imaging can detect transiently appearing residual structures, andtherefore we can select images of IDRs in the fully disordered state. All the log-log plotsof mean 2D end-to-end distance 〈R2D〉 vs. Naa (contained in a fully disordered IDR)

Biomolecules 2022, 12, 1876

5 of 19

domain (1–81) containing a WW domain (48–81) followed by a constantly disorderedregion (82–265). The three structural parameters of PQBP-1 (1–214) were measured frommany images captured at six different imaging rates (6.7–50 fps): the height of the globule(H1 ), the height of the disordered region (H2 ), and the two-dimensional (2D) end-to-end
distance between the N-terminal globule and the C-terminal end (R2D ). The R2D was
calculated as R2D = D − H1 /2 − H2 /2, where D is the direct distance between the Nterminal globule and the C-terminal end. Note that R2D can be measured accurately despite
the fact that all pixel data contained in an image are not acquired simultaneously but atdifferent times due to raster scanning of the sample stage during one-frame acquisition.
This is because the N-terminal globule is nearly stationary, although the C-terminal IDR
moves fast on mica; the positions of the N-terminal globule and the C-terminal end canbe considered as detected at the same time. As shown in Figure 1b–d, the mean values ofthese parameters, (H1 ), (H2 ), and (R2D ), were independent of the imaging rate. Moreover,the displacement of the C-terminal end of WT PQBP-1 in the fast scan X-direction during agiven time interval was measured relative to the N-terminal globule. The displacement
was distributed symmetrically around the zero-mean displacement, irrespective of the timeinterval (Figure 1e). Note that in raster scanning, the sample surface is traced (from –X to+X) and retraced (from +X to –X) in the fast scan direction (Figure 1a), but only images
captured in trace imaging are used. Therefore, the symmetric distribution of the relative
displacement indicates no effect of the tip on the fast motion of the flexible IDR on mica.
The time interval independency of the displacement distribution suggests that the shape of
the IDR changes much faster than the image acquisition rate. The autocorrelation functionof R2D (t) was zero except for ∆t = 0, even when the HS-AFM images were captured at
50 fps [40]. While the tip intermittently contacts the sample, the mica surface is always
in contact with the sample. Therefore, the mica surface may have a non-negligible effect,
especially on the highly flexible structure of IDRs. An IDR is confined to a 2D solution
space on the mica surface, entailing (R2D ) longer than the mean three-dimensional (3D)
end-to-end distance in solution, (R3D ). However, similar confinement can also occur in thecrowded cytoplasm, nucleus, and viral capsid.
4. Constancy of Flexibility/Rigidity of Fully Disordered Regions
Except for rare cases, chemically denatured polypeptide chains are known to haveunique flexibility or rigidity, irrespective of the amino acid sequence and composition [48].
This constancy can be expressed by a power law for the relationship between the numberof amino acids Naa and the mean radius of gyration, hRg i: i.e., hRg i = 0.1927 nm × Naa 0.598(Figure 2a) [48]. In the 3D worm-like chain model for semi-flexible polymer chains, themean square of Rg , i.e., hRg 2 i, can be expressed as a function of the 3D persistence length
P3D as



D E
3
4
2P3D
2P3D
P3D L
L
2
2
Rg =
− P3D +
− 2 × 1 − Exp −
(1)
3
L
P3D
L
where L is the contour length of a polymer chain. In the case of polypeptide chains, L canbe calculated approximately as L = 0.360 nm × (Naa − 1); 0.360 nm is the average distance
between two adjacent amino acids [49,50]. The hRg i data of chemically denatured polypeptide chains approximately followed Equation (1), suggesting that chemically denatured
proteins behave like worm-like chains. From this fitting, the value of P3D is determined tobe 0.976 nm.
In contrast to chemically denatured polypeptides, the SAXS-measured hRg i values of
72 IDRs without permanent secondary structures in the native state have been reported not
to follow a unique power law [51]. However, detailed structural analyses on some of these
IDRs have identified regions that tend to form residual structures [52–55]. In contrast to
SAXS, HS-AFM imaging can detect transiently appearing residual structures, and therefore
we can select images of IDRs in the fully disordered state. All the log-log plots of mean 2D
end-to-end distance hR2D i vs. Naa (contained in a fully disordered IDR) measured by the
HS-AFM imaging of nine different constructs of IDPs were on a single line representing a

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0.517 (Figure 2b) [40]. When worm-like chains freelyi = 1.16 nm
power
lawby
of thehR2DHS-AFM
× Naaofmeasured
imagingnine different constructs of IDPs were on a single
i and wormequilibrateon the asurface
in aof2D
betweenhR2D 2Whenthe 2Dline representing
poweraslaw〈Rsolution,2D〉 = 1.16thenmrelationship× Naa0.517 (Figure2b) [40].
persistence
length(P)
isexpressed
as[56,57]2D
like chains freely equilibrate on the surface as in a 2D solution, the relationship between [56,57] 〈R2D2〉 and the 2D persistence
length(P2D) is expressed
as
D
E
L
2P
R22D = 4LP2D 1 −2𝑃 2D 1 − Exp −𝐿
.
(2)
2D
2 〉
〈𝑅2D
= 4𝐿𝑃2D [1 − L {1 − Exp (− 2P2D)}] . (2)𝐿
2𝑃2D
Because the nine hR2D i data were well fitted to Equation (2), as shown in Figure 2c,
Because the nine 〈R2D〉 data were well fitted to Eq. (2), as shown in Figure 2c, these IDRs
these IDRs in the fully disordered state are uniquely characterized by the measured P2D
in the fully disordered state are uniquely characterized by the measured P2D value of 1.16
value of 1.16 nm. In addition, the fully disordered IDRs are also uniquely characterized
nm. In addition, the fully disordered IDRs are also uniquely characterized by their meanby their mean diameter of approximately 0.5 nm. The unique power law enables thediameter of approximately 0.5 nm. The unique power law enables the determination ofdetermination of Naa contained in a fully disordered IDR from its HS-AFM images, as
Naa contained
described
later.in a fully disordered IDR from its HS-AFM images, as described later.
However,thesubstrate
substratesurface
surfacecan
canhave
additional
effectarising
2D fricHowever, the
have
an an
additional
effectarising
fromfrom
2D frictionaltionalforceslocallyexertingfromthesurfaceagainstthefast
2D
Brownianmotionof aforces locally exerting from the surface against the fast 2D Brownian motionof a highly√
highlyflexible
IDR
chain
[58].
In
the
absenceof
thiseffect,〈R2D〉 is identical to 2√3 × 〈Rg〉,flexible IDR chain [58]. In the absence of this effect, hR2D i is identical to 2 3 × hRg i, whereg〉 is the mean radius of gyration in 3D solution (not on the substrate surface).
hwhereRg i is 〈Rthemean radius of gyration in 3D solution (not on the substrate surface). The
The
comparisonof hour
2D〉 values for the nine different IDP constructs with SAXS-measi values
comparison of our
R2D〈Rfor the nine different IDP constructs with SAXS-measured
ured
〈R
g
〉
valuesoffour
PQBP-1
IDR constructs
andtau protein
constructs
withouthRg i values of four PQBP-1
IDR constructs
and 10tau10protein
constructs
withoutlarge
large
extendeddomains[55]
quantitatively
revealed
that
IDRs
are
swelledbythefrictional
extended domains [55] quantitatively revealed that IDRs
areswelledbythefrictional
force√
force mica
from mica
by a factorof 1.24,= 1.24g.〉.Thus,
Thus,
we
could
derivefromby a factorof 1.24,
i.e., hi.e.,
R2D〈R× 2× 2√3
3 × h×R〈R
i
we
could
deriveaa
i =2D〉1.24
g
0.532
power law
law for h〈RNaaN0.532
and
P
3D =
0.765
nm.
In
literature,
we
can
find
power
Rgg〉i as
as〈RhRgg〉 i==0.248
0.248nm
nm× ×
and
P
=
0.765
nm.
In
literature,
we
can
aa
3D
many
SAXS-measured〈Rg〉 hdata
of IDPs
that that
follow
this thispower
law law[59–69],although
the
find
many
SAXS-measured
Rg i dataof IDPs
follow
power[59–69],althoughabsenceofresidual
structures
in
these
IDPs
is
unknown.
Note
that
the
value
of
P
3D
(0.765the absence of residual structures in these IDPs is unknown. Note that the value of P3Dnm) isnm)
smallerthan thatchemicallydenaturedpolypeptides(0.976nm), nm),indicatingthat(0.765is smaller
thanofthatof chemicallydenaturedpolypeptides(0.976indicatingchemicallydenaturedpolypeptides
are more
extendedthan thanfully fully
disordered
IDRs.IDRs.
This
that
chemicallydenaturedpolypeptides
are more
extendeddisordered
distinctdifference
can beexplained
simplyby thefactfact
thatthat
denaturants
such
as
This
distinctdifference
canarguably
be arguably
explained
simplyby thedenaturants
such
urea
and
guanidinium
chloride
arearegood
solvents.
Therefore,
the thepolypeptide
chain
is exasurea
andguanidinium
chloride
good
solvents.
Therefore,
polypeptide
chain
isextendedbecauseinteraction
betweenthe
chain
backbone
anda goodsolvent
solventisisfavorafavortended because
thetheinteraction
betweenthe
chain
backbone
anda goodable.
contrast,chain-chain
chain-chaininteractions
interactionsare
arerepulsive,
repulsive, althoughalthough different
different interpretations
ble. InIncontrast,
may be possible [70].

Figure 2.
molecular
dimensions
andandthe thenumberof aminoacid residues.
(a,b)
Figure2. Relationship
Relationshipbetween
betweenmolecular
dimensions
numberof aminoacid residues.
Power laws are found in chemically denatured proteins (a) and fully disordered regions within IDPs(a,b) Power laws are found in chemically denatured proteins (a) and fully disordered regions within(b). The data in (a) are from the SAXS study by Kohn et al. [48]. The mean R2D values, 〈R2D〉, in (b)IDPs (b). The data in (a) are from the SAXS study by Kohn et al. [48]. The mean R2D values, hR2D i, inare those for fully disordered IDRs observed in the indicated protein constructs. (c) The mean square(b)those for fully disordered IDRs observed in the indicated protein constructs. (c) The meanof Rare2D values, 〈R2D2〉, of the nine IDP constructs shown in (b) are fitted to Eq. (2), from which the
2 i, of the nine IDP constructs shown in (b) are fitted to Equation (2), fromsquarehR2Dvalue ofofPR2D2Disvalues,
estimated
to be
1.16 nm. The number of residues in (b) is those contained in the fully
which
the value
disordered
IDRs.of P2D is estimated to be 1.16 nm. The number of residues in (b) is those contained in
the fully disordered IDRs.

5. IDPs with Dumbbell Shape
5. IDPs with Dumbbell ShapeIDPs randomly selected for HS-AFM imaging often showed a dumbbell-like shape
IDPs randomly selected for HS-AFM imaging often showed a dumbbell-like shape (i.e.,(i.e., two structured domains are linked via an IDR): e.g., the archaeal Hef (helicase-assotwo structured domains are linked via an IDR): e.g., the archaeal Hef (helicase-associatedciated endonuclease for fork-structured DNA) protein [38], bacterial FliK protein [39] andendonuclease for fork-structured DNA) protein [38], bacterial FliK protein [39] and yeast
yeast Atg1 [40] (Figure 3a–c). In addition, other methods found various dumbbell-shaped

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7 of 18

Atg1 [40] (Figure 3a–c). In addition, other methods found various dumbbell-shaped
IDPs: e.g., the regulatory subunits of the cAMP-dependent protein kinase [71], cardiac
IDPs: e.g., the regulatory subunits of the cAMP-dependent protein kinase [71], cardiac
troponin C [72], the DNA repair factor CtIP [73], CAHS (cytoplasmic abundant heat solutroponin C [72], the DNA 2+repair factor CtIP [73], CAHS (cytoplasmic abundant heat soluble)ble) protein [74], and Ca -unbound calmodulin [75]. In these IDPs, the N-terminal domain
protein [74], and Ca2+ -unbound calmodulin [75]. In these IDPs, the N-terminal domain(NTD) and the C-terminal domain (CTD) often possess distinct functions. For example,(NTD) and the C-terminal domain (CTD) often possess distinct functions. For example,
Hef has a helicase domain at the N-terminus and a nuclease domain at the C-terminus
Hef has a helicase domain at the N-terminus and a nuclease domain at the C-terminus
and dimerizes via the nuclease domain. The helicase activity is dramatically stimulated
and dimerizes via the nuclease domain. The helicase activity is dramatically stimulated by
by fork-structured
DNAs,
the nuclease
specificallycleaves
nicked,flapped,andfork-structured
DNAs,
whilewhile
the nuclease
specificallycleaves
nicked,flapped,and forkfork-structured
Fromactivities
these activities
and phenotype
ofstrains,mutantstructured
DNAsDNAs[76,77].[76,77].
From theseand phenotype
analysesanalysesof mutantstrains,Heffunctionsmainlyinstalledreplication
fork
repair
[78].
In
the
case
of
Atg1,
the
Hef functions mainly in stalled replication fork repair [78]. In the case of Atg1, the solesolekinaseinall
Atgproteins,
the
NTD
is
a
kinasedomain,
while
the
CTD
is
the
tandemkinase in all Atg proteins, the NTD is a kinase domain, while the CTD is the tandem MIT
MIT (microtubuleinteracting
and transport)
thatthebinds
the MIT-interacting
motif(microtubuleinteracting
and transport)
domaindomainthat binds
MIT-interacting
motif (MIM)(MIM)
in
Atg13.
The
NTD
in
FliK
works
as
a
lengthrulerofthebacterial
flagella
hook,in Atg13. The NTD in FliK works as a length ruler of the bacterial flagella hook, while thewhile
the CTDtofunctions
to switch
secretionfromthe hook
proteinto thefilament
CTD
functionsswitch secretionfromthe hook
proteinto thefilament
proteinin theprotein
typeinthe
type-III
flagellar
exportapparatus[79].
The
domaindiagramsofthese
dumbbellIII flagellar export apparatus [79]. The domain diagrams of these dumbbell-shaped
IDPs
N fragment
shapedare shownin FigureNMR
3d. Aanalysis
previous
NMR
analysisof theFliKNindicatedfragment
areshownIDPs
in Figure
3d. A previousof theFliK(1–147)
C fragment (204–370) isC(1−147)indicatedalargely
unfoldedstructure,
whereastheFliK
a largely unfolded structure, whereas the FliK fragment (204–370) is structured, except forstructured,its N-terminal
204–252[80].
The HS-AFM images
dumbits
N-terminalexcept
regionfor
204–252[80]. Theregion
HS-AFM
imagesof dumbbell-shaped
FliKofshowedbell-shaped
showedlargerheight,nm) andsmaller(meanconnectedheight, 2.0bynm)
larger(mean FliK
height,
2.7 nm)and(meansmaller(mean2.7
height,2.0 nm)globulesa
globulesconnected
a flexible
linker (Figure
3c).
HS-AFM
imagesof deletion
conflexible
linker(Figureby
3c).
From HS-AFM
imagesof Fromdeletionconstructsof FliK,the larger
structsof FliK,the larger
and smaller
globuleswereidentified
as the[39].
NTD
and CTD,
reandsmaller
globuleswere identified
as theNTD and
CTD,
respectively
However,theirspectively[39].
However,their
size
differenceisinconsistent
withthedomainarrangesize difference is inconsistent with the domain arrangement in FliK (Figure 3d), suggestingment
FliKis(Figure3d), suggestingthat
is loosely
folded.
There may
be twothat
theinNTD
loosely folded.
There maybe thetwoNTDpossibilities
for thedisagreement
betweenpossibilities
theresults
disagreement
betweenofHS-AFM
results
ofstructure
the structure
HS-AFMand forNMRof the structure
FlikN ; (i)and
the NMRlooselyfoldedof theof
N fragment could not be detected but was
FlikNN fragment; (i) the loosely
structurebutof theFliK
could folded
not be detected
wasFliK
detectedby NMR as unstructured, or (ii) theN fragment
detectedby NMRas unstructured, or (ii) the FlikN fragment is unfolded.
Flik
is unfolded.

Figure 3. Dumbbell-shaped IDPs observed by HS-AFM. (a–c) Clips from HS-AFM images and their
Figure 3. Dumbbell-shaped IDPs observed by HS-AFM. (a–c) Clips from HS-AFM images andschematized molecular features of the three IDPs. The light blue arrowheads point to IDRs. (d) Dotheir schematized molecular features of the three IDPs. The light blue arrowheads point to IDRs.
main diagrams of the three IDPs. (e) Effective concentration of CTD (NTD) around NTD (CTD) as a(d)
Domainofdiagramsof theEffective inconcentrationof CTD (NTD)around NTD(CTD)
function
the numberof threeaminoIDPs.
acids(e)contained
the fully disordered
IDR connecting
NTD
andasCTD.
a function of the number of amino acids contained in the fully disordered IDR connecting NTD
and CTD.

The common structural feature in dumbbell-shaped IDPs must have multiple funcThe common structural feature in dumbbell-shaped IDPs must have multiple functional benefits. In the repair of stalled replication fork by Hef, the fork structure has to betional benefits. In the repair of stalled replication fork by Hef, the fork structure has to bealtered by the helicase, and DNA segments have to be removed by the nuclease. Althoughaltered by the helicase, and DNA segments have to be removed by the nuclease. Althoughother proteins are also involved in the repair process, it is evident that the helicase andother proteins are also involved in the repair process, it is evident that the helicase andnuclease activities must proceed in harmony. The flexible IDR linker connecting the helnuclease activities must proceed in harmony. The flexible IDR linker connecting the helicase
icase and nuclease domains can localize these functional sites more efficiently and faciliand nuclease domains can localize these functional sites more efficiently and facilitates theirtates their harmonized actions compared to the case where separated helicase and nucleharmonized actions compared to the case where separated helicase and nuclease proteinsase proteins work independently. In the case of Atg1, its C-terminal MIT1/MIT2 domain
can bind the dephosphorylated MIM domain located in the C-terminal disordered region

Biomolecules 2022, 12, 1876

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work independently. In the case of Atg1, its C-terminal MIT1/MIT2 domain can bindthe dephosphorylated MIM domain located in the C-terminal disordered region of Atg13.
Therefore, starvation-induced dephosphorylation of Atg13 triggers its binding to Atg1 andthen recruits Atg17-29-31 and Atg11 to form the Atg1 complex (Atg1-Atg13-Atg17-Atg29Atg31) that works as a scaffold for PAS formation [14,26]. The Atg13-Atg17 interaction canoccur when regions close to the MIM domain are dephosphorylated. Although the reaction
timing after PAS or autophagosome formation is not well known, Atg1 kinase phosphorylates Atg13 and Atg11 [27,81], which is considered to dissolve a large complex formed by
the PAS complex and other Atg proteins, and regulates selective autophagy [81,82]. WhenAtg1 phosphorylates the Atg13 IDR by the N-terminal kinase, Atg1 also grasps this IDR
using the C-terminal MIT1/MIT2 domain. Thus, the N- and C-terminal domains of Atg1
need to get in close proximity to each other, which can be facilitated by the long flexible
linker connecting the two interaction sites.
Another functional benefit of the dumbbell-like structure is that this structure allows and facilitates dynamic interactions between the two distantly separated functional
domains. Even when the Kd of intramolecular interactions between distantly separated
domains is large, the effective concentration (Ceff ) of one domain around the other canbe significantly high, enabling their frequent interactions. The Ceff can be estimated as afunction of h R3D i, as follows. A fully disordered IDR is considered to behave as a worm-likechain. In this case, the distribution function of R3D is given by3/2

P( R3D )dR3D = 4πR23D 3
DE
2π R3D 2




2
R
3
3D
E dR3D .
exp− D
2 R 2

(3)

3D

The two globular domains linked via such an IDR associate with each other when
they approach within a certain range of distance. However, when we consider the actual
dimensions of the two globules, R3D becomes a complex function of the distance between
the two globules. To avoid this complexity, we assume that NTD-CTD association occurs
when R3D becomes R0 or shorter (i.e., R3D ≤ R0 ). In this approximation, the probability offinding the CTD in the volume (R3D ≤ R0 ) around the NTD is given by
p=

Z R0
0

P( R3D )dR3D(4)

Therefore, the Ceff of the CTD in the volume in close proximity to the NTD is given as
4π 3Ceff = α × p × 10−3 /R0 · NA(5)3where NA is the Avogadro constant and α is a steric hindrance effect factor by which the
NTD–CTD interaction frequency is reduced. Considering the NTD surface available for
CTD binding, the steric hindrance factor is approximately 0.5. Supposing that the IDR
contains 100 amino acids, its h R3D i value is 7.25 nm, under which Equation (4) provides
p = 3.56 × 10−3 . Therefore, the effective concentration of the CTD around the NTD isapproximately estimated to be Ceff = 0.71 mM (the Naa dependence of Ceff is shown in
Figure 3e). Thus, the two terminal domains in a dumbbell-shaped IDP can have a large
chance to interact with each other even when their Kd value is in the order of mM.
So far, only 2–3 IDPs with a property of “conformational malleability” have beenfound [26]. Atg13 is such a rarely found IDP. Although it is not dumbbell-shaped, its
N-terminus folded HORMA domain (1–267) interacts with the C-terminus end region of
the long IDR (268–740). The HORMA domain binds Atg9, a membrane protein existingon specific cytoplasmic vesicles (Atg9 vesicles) [83]. The C-terminal IDR contains theAtg1 binding site (460–521) and Atg17 binding sites (359–389 and 424–436) [27]. The
folded HORMA domain becomes largely disordered when the C-terminus is fused tomaltose-binding protein (MBP) [26,27]. The MBP-fusion impairs the PAS localization of

Biomolecules 2022, 12, 1876

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Atg9 in the cell. These observations indicate that transient NTD-CTD interactions are
required for the formation of the stable and functional HORMA structure because the MBP
moiety can interfere with NTD-CTD interactions. The stabilized structure of HORMA
can be retained for a while even after the transient interaction is completely removed(i.e., malleability). Therefore, only occasional interactions are enough to keep the stableand functional HORMA structure. And yet, the functional role of this conformationalmalleability is still unknown. Although not yet clearly demonstrated, Flik possibly exhibits
conformational malleability. That is, when the FlikN -FlikC interaction in cis is lost for acertain time duration, the FlikN domain (1–147) becomes fully disordered. When thisdomain is fully disordered, its contour length becomes ~53 nm, nearly identical to themaintained hook length of the bacterial flagellum (~55 nm), and thus, the FlikN domain
can work as a ruler of hook length. Namely, the N-terminal end of FliK binds to the tip of
the growing hook, while the disordered FliKN chain is moderately stretched in the narrow
channel of the filament. When the hook grows up to ~55 nm, the FlikN chain is fully
stretched and, thus, pulls the FlikC domain remaining in the cytoplasm. This pulling forcechanges the FlikC -secretion gate interaction to switch the secretion substrates from earlyproteins, including the hook protein, to late proteins, including the flagellin, as advocatedpreviously [79,84].
6. Measles Virus IDPs
Before describing the HS-AFM study on two IDPs of measles virus, here I summarize their background. Comprehensive reviews on these IDPs and their functional roles
are available elsewhere [85–88]. The measles virus is a nonsegmented, single-stranded,negative-sense RNA virus. Therefore, after virus entry into host cells, mRNAs coding
for eight different virus proteins with six genes need to be synthesized from the genome
RNA by using its own RNA-dependent RNA polymerase complex comprising the Large(L) protein and its cofactor, the phosphoprotein (P). This synthesis mode is later switchedto the processive mode to produce cRNA for genome replication. The genome RNA is
encapsidated by the nucleoprotein (N) to form a helical nucleocapsid with a diameterof ~18 nm and a helical pitch of ~6 nm, comprising 12.3 protomers per turn of the helix(Figure 4a), as suggested from the cryo-EM analysis of nucleocapsid-like particles [89].
This ribonucleoprotein complex is the substrate for both transcription and replication; thepolymerase complex (L-P) cartwheels on the nucleocapsid template in these processes(Figure 4a). Each N binds six nucleotides, and therefore, the genome length (15,894 nucleotides) is exactly 6 × integer (2649). Monomeric N also exists in the cell as a soluble
monomer which is referred to as N0 , while the assembled form is referred to as NNUC .
N0 requires its chaperone, whose role is played by P and the V protein, which shares thefirst 231 amino acids with P [88]. The binding of P (and also V) blocks the polymerizationof N. The tetrameric P tethers L onto the nucleocapsid template (Figure 4a). Both the N
and P proteins contain large, disordered regions (Figure 4b). The N protein consists of
the N-terminal structured domain (Ncore : 1–400) and the C-terminal disordered domain(NTAIL : 401–525). Note that the aforementioned nucleocapsid-like particles are formed
without NTAIL ; in the presence of NTAIL , the nucleocapsid is very flexible. The P protein
consists of the N-terminal domain containing a large, disordered region (PNT: 1–230)
and the C-terminal domain (PCT: 231–507) (Figure 4b). Transcription requires only PCT,
whereas genome replication also requires PNT. The N-terminal PNT moiety (PN37 : 1–37)
binds N0 , while the PCT region (304–376), referred to as PMD, is responsible for coiled-coiloligomerization of P and binding to L, and the C-terminal end region (459–507) referred toas the X domain (XD) is responsible for binding to NTAIL (the segment PLoop connecting
PMD and XD is disordered). Thus, the formation of the N0 -P complex is mediated by
the dual PNT-NCORE and PCT-NTAIL interaction. In a different description of the domainorganization of P, the region 1–304, larger than PNT, is referred to as PTAIL , which is mostly
disordered except for PN37 [88].

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10 of 18

Figure 4. Measles virus IDPs, N and P. (a) Schematic showing complexes of N0–P, NNUC–P, and L–P
Figure 4. Measles virus IDPs, N and P. (a) Schematic showing complexes of N0 -P, NNUC -P, and L-P
RNA polymerase. The location of RNA is schematically represented for its visualization. Only a partRNA
The location of RNA is schematically represented for its visualization. Only a partof Npolymerase.
TAILs is shown. (b) Domain diagrams of N and P proteins. The light-color regions indicate IDRs.
of(c)
NTAIL
s is shown.
(b) Domain
diagramsfromof Ntheandgroove
P proteins.
The light-color
regions indicate IDRs.
Schematic
showing
NTAIL extendingof nucleocapsid
template.
(c) Schematic showing NTAIL extending from the groove of nucleocapsid template.

There are fundamental questions to be solved about the measles viral transcription
are fundamental
questions
to(i)
behow
solvedabout
the measlesswitch
viral transcription
andThere
replication
processes. For
example,does
the polymerase
between tranand replication processes. For example, (i) how does the polymerase switch between transcriptive and replicative modes?, (ii) how are viral and cellular RNAs discriminated byscriptive and replicative modes? (ii) how are viral and cellular RNAs discriminated by thethe N protein?, (iii) how can the L-P complex access the RNA which winds around the
N protein? (iii) how can the L-P complex access the RNA which winds0around the groove
groove of the helical polymer of N protein?, and (iv) why does the N –P complex need toof the helical polymer of N protein? and (iv) why does the N0 -P complex need to be formed
be formed before the N protein polymerizes? Various attempts to answer these questions
before the N protein polymerizes? Various attempts to answer these questions and others
and others have been carried out based on the structural features of N and P and their
have been carried out based on the structural features of N and P and their interactions.
interactions. Some of the mechanistic proposals that answer these questions are summaSome of the mechanistic proposals that answer these questions are summarized here. For
rized here. For more details, see reviews [85–88]. For instance, it has been proposed that
more details, see reviews [85–88]. For instance, it has been proposed that the mode switch
the mode switch between transcription and replication is due to a conformational change
between transcription and replication is due to a conformational change of the nucleocapsidof the nucleocapsid template caused by the folding of NTAIL induced by the interaction
template caused by the folding of NTAIL induced by the interaction with XD [85]. However,with XD [85]. However, a completely different mechanism has also been proposed for aa completely different mechanism has also been proposed for a negative-sense RNA virus
negative-sense(influenza
Thatis, there
be no
mode
but
(influenza
virus).RNA
Thatvirus
is, there
may bevirus).
no modeswitch,butmaynascentcRNA
is switch,degraded
nascentcRNA
isdegraded
by
host
cell
nucleases
unlessitisstabilizedby
newly
syntheby host cell nucleases unless it is stabilized by newly synthesized N and P [90]. NTAIL -XD
sized Nisand[90]. NTAILto–XD
bindingis also suggested
towithassist
L nucleocapsid
in remaining templatein contact
binding
alsoPsuggested
assist
L in remaining
in contact
thewith thenucleocapsid
templateduring [91].
successivesynthesisof mRNAs[91]. The binding0
duringsuccessivesynthesisof mRNAs
The bindingof P
N37 (and also VN37 ) to N
of
P
N37 0
(and
also
V
N37) to N0 keeps N0 in an open conformation ready to grasp viral RNA
keeps N in an open conformation ready to grasp viral RNA and is suggested to avoid
and is suggested
toof
avoid
of Nisto
cellular RNA[92].theNTAIL
is sugnon-specificbinding
N to non-specificcellular RNAbinding[92]. NTAIL
suggested
to loosen
helically
gestedtoloosenthehelically
assembledribonucleoprotein
complex,facilitatingtheassembled ribonucleoprotein complex, facilitating the transcription and replicationtranby
scription
L-P[93]. and replication by L–P [93].
HS-AFMStudy
StudyononNN
TAIL
7.7.HS-AFM
TAIL

Prior
thetheHS-AFMstudy
on N(401–525)[40],[40],its structuralfeatureshave alreadyPriortoto
HS-AFM
study
onTAILNTAIL(401–525)
its structuralfeatureshave albeenwellcharacterized
quantitatively
byvariousmethods.
The
tworegions,
calledready been well characterized quantitatively by various methods. The two regions, Box1
called(401–420)
and Box2
were previously
shown
to containa linearmotif
withwith
αBox1 (401–420)
and(486–502),
Box2 (486–502),
were previously
shown
to containa linearmotif
helicalpropensity(α-MoRE)(Figure4c)[93,94]
undergoingtransitions
amongcompletely
α-helical propensity (α-MoRE) (Figure 4c) [93,94] undergoing transitions among comunfoldedand folded
helicalconformations[95–99]. The
NMR-detected
unfoldedunfolded
state ofpletely unfolded
andfoldedhelical conformations[95–99].
The NMR-detectedBox2
isshort-lived(µsorder)[86].
Thebinding
of
Box2
to
XD
in
a
tripleα-helical
bundlestate of Box2 is short-lived (μs order) [86]. The binding of Box2 to XD in a triple α-helicalisbundlesuggested
to form a toquadruple
α-helical bundle[100].
Box1[100].
is locatedgrooveis suggested
form a quadruple
α-helical
bundleBox1inis thelocatedin oftheNUC (Figure 4c) [89,93]. In addition to Box1 and Box2, the region (517–525) is called Box3
Ngroove
NUC
of N(Figure 4c) [89,93]. In addition to Box1 and Box2, the region (517–525) is
becauseit is suggested
XD [101].
However,
Box3However,is not considered
α-MoRE
becauseαcalled Box3
because ittoisbind
suggested
to bind
XD [101].
Box3 is notconsidered
the secondary structure stabilizer, 2,2,2-trifluoroethanol (TFE), does not promote α-helical
MoRE because the secondary structure stabilizer, 2,2,2-trifluoroethanol (TFE), does notfolding within Box3 [101], unlike Box1 and Box2. Box3 possibly remains disordered evenpromote α-helical folding within Box3 [101], unlike Box1 and Box2. Box3 possibly remains
after binding to XD. Thus, the regions containing 88 amino acids in the two IDRs (421–485
disordered even after binding to XD. Thus, the regions containing 88 amino acids in theand 503–525) are considered to be fully disordered. Since most of these structural features of
two IDRs (421–485 and 503–525) are considered to be fully disordered. Since most of thesestructural features of NTAIL are already established, the HS-AFM imaging of NTAILBiomolecules 2022, 12, 1876

11 of 19NTAIL are already established, the HS-AFM imaging of NTAIL constructs aimed to examine
whether HS-AFM could faithfully detect these known structural features of NTAIL andto determine dynamic structural transition rates that could not have been analyzed withother methods. As already included in Figure 2b, hR2D i = 11.6 ± 4.4 nm measured with
the HS-AFM imaging of NTAIL -GFP fusion (Figure 5a) approximately followed the powerlaw, hR2D i = 1.16 nm × Naa 0.517 (GFP was used to impede the otherwise very fast diffusionof NTAIL on mica). In this fusion construct, Box1 was always visible, whereas Box2 only
occasionally appeared because it is positioned close to the large GFP moiety. The height
distribution of Box1 was well fitted to a double Gaussian function with peaks at 0.8 nm
and 1.1 nm (Figure 5b). The former is larger than the height of fully disordered regions(~0.5 nm), while the latter is identical to the diameter of a single α-helix. The area ratioof the two Gaussian components (Ke = higher state/lower state) was 0.28, indicatinga propensity of Box1 to take partially disordered conformations. To observe dynamic
structural transitions of Box2, NTAIL was fused to thioredoxin (Trx) at the N-terminus of
NTAIL (Figure 5c). The height distribution of Box2 was also well fitted to a double Gaussianfunction with peaks at 0.8 nm and 1.1 nm (Figure 5d). However, Box2 tends to form an
α-helix compared to Box1, judging from its larger Ke value, 1.91. From these observations,
we conclude NTAIL is fully disordered except for Box1 and Box2. Since the measured R2Dvalues are widely distributed due to the highly flexible nature of the disordered regions,changes of R2D resulting from the structural transitions of small Box1 and Box2 are masked
by this wide distribution of R2D and their preferred tendencies to take partially disordered
and fully folded states, respectively. Nevertheless, the time-varying R2D values are wellcorrelated with the time-varying height values of Box1 and Box2, suggesting that the IDR
length partially changes due to the transitions in these globules between the partially andfully folded states. One of the advantages of HS-AFM imaging of IDPs over other methods
is that we can directly observe structural transitions and, therefore, determine the transition
rates unless they are faster than ~50 s−1 . To determine the transition rates of Box1 and Box2,
the autocorrelation functions G(τ) of their time-series height data H(t) were first calculated(Figure 5e,f) as
N −h

N

n =1

n =1

G (τ ) ≡ ∑ [ H (n∆t) − µ̂][ H ((n + h)∆t) − µ̂]/ ∑ [ H (n∆t) − µ̂]2(6)where τ ≡ h∆t (h = 0, 1, 2, 3, . . . , N–1) and µ̂ is the mean value of H(n∆t) (n = 1, 2, 3, . . . , N).
When the order-to-disorder and disorder-to-order transition rates are expressed by kOD andkDO , respectively, the G(τ) relaxation rate denoted by λ is expressed as λ = kOD + kDO . From
the relationship, Ke = kDO /kOD , kOD and kDO can be expressed as functions of λ and Ke askOD = λ/(1 + Ke ) and kDO = λKe /(1 + Ke ). In this way, the rate constants could be determined
as kOD = 5.7 s−1 and kDO = 1.6 s−1 for Box1 and kOD = 1.1 s−1 and kDO = 2.0 s−1 for Box2.
Thus, the dynamic structural features of NTAIL are quantitatively delineated, as shown in
Figure 5g. In both Box1 and Box2, the structural transitions are relatively slow, comparable
to the elongation rate of the L-P polymerase (three nucleotides/s) [102]. Although this ratecomparability may be just a coincidence, NTAIL has to change its structure in concert withthe motion of the L-P polymerase cartwheeling on the nucleocapsid template. For example,
Box2 has to change its affinity for P (at XD) during this L-P polymerase motion. In contrast,
Box1 has to be either disordered or folded (but likely the former) to loosen the helicalnucleocapsid structure by interfering with the stacking of turns (see Figure 4a,c) [93,103] sothat the polymerase reaction is facilitated.

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Figure 5. HS-AFM analysis of structure and dynamics of NTAIL. (a) HS-AFM images and their scheFigure 5. HS-AFM analysis of structure and dynamics of NTAIL . (a) HS-AFM images and theirmatics of NTAIL-GFP. The open arrowheads point to the N-terminal end of NTAIL. (b) Height distrischematics
of N-terminal
NTAIL -GFP.globule
The open
point tothe N-terminalend of NTAIL. (b) TAIL
Heightbution of the(i.e.,arrowheadsBox1). (c) HS-AFM
imagesand their schematics
of Trx-N.
distributionof the N-terminal(i.e., Box1).
(c) HS-AFM
imagesanddistribution
their schematics
The open arrowheads
point toglobule
the C-terminal
globuleof NTAIL. (d)Heightof the ofC- TrxglobuleBox2). (e,pointf) Autocorrelation
functionsof time-series
heightdata distributionof Box1 (e) of
Nterminal
open (i.e.,
arrowheads
to the C-terminal
globuleof NTAIL . (d)HeightTAIL . The
andC-terminal
Box2 (f). The
valuesof λBox2).
represent
respective decay
constantsof the autocorrelation
theglobule(i.e.,(e,f) the
Autocorrelation
functionsof time-series
height datafuncof Box1
. The topof
andbottompanelstions.
Schematics
structuraland dynamic
featuresdecayof NTAIL(e)and(g)
Box2(f). Theshowing
values of
λ represent
the respective
constants
theautocorrelation
correspond to the more- and less-ordered states, respectively. The red arrows indicate the kinetic
functions. (g) Schematics showing structural and dynamic features of NTAIL . The top and bottom
nature of changes in height of Box1 and Box2.
panels correspond to the more- and less-ordered states, respectively. The red arrows indicate thekinetic nature of changes in height of Box1 and Box2.

8. HS-AFM Study on PNTWhen we started the HS-AFM imaging of PNT in 2012, nothing was known about its
structure except for the resistance to proteolysis of the N-terminal 27–99 region of PNT in thepresence of TFE [104]. In 2016, a crystal structure was revealed for a fused protein complex,
P1–48 -N0 21–408 , that is devoid of the disordered regions of P and N [105]. This chimera
protein is dimerized in the crystal, and P1–48 forms two α-helices (longer α1 and shorterα2) connected with a short loop. The α1 and α2 bridge two adjacent N0 21–408 protomers,
although this arrangement in the chimera proteins may not occur in the physiological case.
It is also uncertain whether the N-terminal region, P (1–48), forms α-helices without N0 .
In 2018, the Blackledge group reported the NMR structures of PTAIL (1–304) alone andthe complex between the full length of N and PTAIL [106]. In the NMR structure of PTAIL
alone, four regions were identified to have propensities to form α-helices and thus named
α1/2 (1–37), α3 (87–93), and α4 (189–198). These three regions are linked by highly flexible
IDRs. The IDR segment (125–170) that links α3 and α4 is rich in acidic residues and devoidof basic residues and, therefore, referred to as the acidic loop. In the N0 -PTAIL complex
structure, a region containing α4 (unstructured 181 DVETA185 termed δ + α4) was identified
8. HS-AFM Study on PNTas an additional N interaction site. This interaction occurs independently of the presence of
we startedin
theimagingof PNTin 2012,interaction
nothing was
known
abouttoitsbe aα1/2. WhenIts counterpart
NHS-AFM
responsible
for this
additional
was
identified
structure
exceptfortheresistancetoproteolysisoftheN-terminal
27–99
regionof
PNT
incontiguous region 96–127 in NCORE . This interaction occurs transiently with a time constantthepresenceofTFE[104].
In2016,
acrystalstructurewasrevealed
for
a
fused
proteinin the order of ms and Kd = 0.6 mM. Although this Kd is large, the effective concentration
P1–48–N021–408, that is devoid of the disordered regions of P and N [105]. This chiCcomplex,
eff of δα4 around the NCORE can be significantly enhanced by the α1/2-NCORE and NTAIL mera protein is dimerized in the crystal, and P1–48 forms two α-helices (longer α1 and
XD associations and the high flexibility of the linker segments, as mentioned0 in Section 5.
shorter α2) connected with a short loop. The α1 and α2 bridge two adjacent N 21–408 proAn essential role of this δα4–NCORE interaction in the polymerase reaction was shown by atomers, although this arrangement in the chimera proteins may not occur in the physiotranscription assay. Since α3 and the acidic loop are free from the other part of PTAIL andlogical case. It is also uncertain whether the N-terminal region, P (1–48), forms α-helices
not bound toN, the highly flexible motion of the long flexible segment between α1/2
without N0. In CORE
2018, the Blackledge group reported the NMR structures of PTAIL (1–304)
and δα4 dynamically occupies a large space around NCORE . This large space occupation
alone and the complex between the full length of N and PTAIL [106]. In the NMR structure
potentially inhibits the interaction of RNA, N monomers, and other proteins with NCORE ,as discussed in this study [106]. As demonstrated in the HS-AFM study on a yeast prion
protein Sup35, the highly flexible motion of long IDRs can have such a repelling effect [43].

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However, despite these structural details revealed by the NMR analysis, the resistance of
the N-terminal 27–99 region of PNT to proteolysis cannot be rationalized from the NMR
data, suggesting the existence of a large-scale structure undetectable by NMR.
As described below, the combination of the NMR structure of PTAIL and results fromthe HS-AFM imaging of PNT (1–229)-GFP fusion enriched our understanding of the
dynamic structure of PNT and its functional relevance. The HS-AFM images of PNT-GFP(Figure 6a) showed an N-terminal globule connected to the GFP moiety via a flexible
chain undergoing length changes. In addition, a small globule close to GFP could beoccasionally detected, probably corresponding to α4. The mean height of the N-terminal
globule remained constant at 1.1 nm (Figure 6b) while its lateral dimensions varied. The
height of 1.1 nm corresponds to the diameter of a single α-helix. Therefore, the N-terminal
globule contains α1/2 (and possibly also α3) as the highest particle and other regions lower
than α1/2. The R2D value of the flexible chain connecting the GFP and the N-terminal
small globule is distributed widely. Its distribution was well fitted to a double Gaussianfunction with peaks at 8.9 and 14.3 nm (Figure 6c). The mean height of the flexible chain
was 0.4–0.5 nm in both metastable shorter and longer states. This suggests that the flexible
chain is always fully disordered in both metastable states, except for the occasionallyappearing α4 (α3 did not appear in the HS-AFM images as a distinct region). From thepower law, hR2D i = 1.16 nm × Naa 0.517 , the Naa contained in the shorter and longer chainswere estimated to be 51 and 128, respectively. The presence of small α3 and α4 regionsthat may be undergoing structural transitions does not much affect this estimation. Fromthese Naa values, the number of amino acids contained in the N-terminal small globule
was approximately estimated to be 178 and 101 for the shorter and longer chain states,
respectively. From the number 101, the N-terminal small globule always contains α1/2
(1–37), α3 (87–93), and an IDR segment (38–86) connecting α1/2 and α3. Therefore, theseregions always form a compact structure, explaining no appearance of α3 as a distinct region
in the HS-AFM images. Importantly, this compact structure also explains the resistance toproteolysis of the N-terminal segment (27–99) containing the fully disordered IDR segment(38–86). The number 178 indicates that the structural transitions of PNT occur on a large
scale, mainly by association and dissociation between the N-terminal compact structure
and the acidic loop (125–170). From the degree of order propensity, Ke = 3.73, the larger
compact structure, including the acidic loop, is formed more frequently than the smaller
compact structure without the acidic loop. The kOD and kDO values of the observed global
structural transitions were estimated as kOD = 0.5 s−1 and kDO = 2.0 s−1 from the correlation
function of time-series R2D data (Figure 6d) and the Ke value. Thus, the dynamic structuralfeatures of PNT are quantitatively delineated, as shown in Figure 6e. These rates areagain comparable to the elongation rate of the L-P polymerase. In the larger compact
form, δα4 cannot reach its interaction site on NCORE . α1/2 also may not be available for
NCORE binding. Since the δα4-NCORE interaction is essential for RNA transcription, thedocking and undocking of the acidic loop and the N-terminal structural unit (i.e., thesmaller compact structure) must be regulated at certain steps in the transcription process.
The cartwheeling L-P around the nucleocapsid may pull the acidic loop for its undocking,enabling smooth transcription. Since the N-terminal smaller and larger compact structures
are not detected in the NMR analysis, some of the structural and dynamic features in the
N0 -P complex model constructed from NMR data have to be amended. However, thiscomplex also needs to be investigated by using HS-AFM.

Biomolecules 2022, 12, 1876

matics of NTAIL-GFP. The open arrowheads point to the N-terminal end of NTAIL. (b) Height distribution of the N-terminal globule (i.e., Box1). (c) HS-AFM images and their schematics of Trx-NTAIL.
The open arrowheads point to the C-terminal globule of NTAIL. (d) Height distribution of the Cterminal globule (i.e., Box2). (e, f) Autocorrelation functions of time-series height data of Box1 (e)and Box2 (f). The values of λ represent the respective decay constants of the autocorrelation functions. (g) Schematics showing structural and dynamic features of NTAIL. The top and bottom panels
14 of 19
correspond to the more- and less-ordered states, respectively. The red arrows indicate the kinetic
nature of changes in height of Box1 and Box2.

8. HS-AFM
Studyon PNT
Figure6. HS-AFM
analysisof structure and dynamics of PNT. (a) HS-AFM images and their schematWhen we The
startedthe
HS-AFM imaging
of PNTin 2012,globulenothing
about
its
ics of PNT-GFP.
open
arrowheads
point to the
N-terminalof was
PNT,known
while thelight-blue
structure
exceptfortheresistancetoproteolysisoftheN-terminal
27–99
regionof
PNTarrowhead points to a small globule appeared in the middle of disordered region (very likelyin
correthe presence
of(b)
TFE
[104].distribution
In 2016, a of
crystalstructureglobule.
was revealedfor 1.1
a fusedsponding
to α4).
Heightthe N-terminal
The value
nm is protein
identical to
021–408, that is devoid of the disordered regions of P and N [105]. This chicomplex,
P1–48
the
diameterof –Na singleα-helix. (c) Distribution of end-to-end distance of fully disordered IDR (R2D ).
meraprotein is dimerized
the crystal,
1–48 forms two α-helices (longer α1 and(d)
Autocorrelation
function ofintime-series
dataandof RP
2D . The value of λ represents the decay constantshorter α2) connected with a short loop. The α1 and α2 bridge two adjacent N021–408 proof the autocorrelation function. (e) Schematics showing structural and dynamic features of PNT. The
tomers, although this arrangement in the chimera proteins may not occur in the physiotop and bottom panels correspond to the more- and less-ordered states, respectively. The numbers inlogical case. It is also uncertain whether the N-terminal region, P (1–48), forms α-helices
red and blue0 represent the numbers of amino acids contained in the respective globules and fully
without N . In 2018, the Blackledge group reported the NMR structures of PTAIL (1–304)
disordered IDRs, respectively, estimated by using the power law and measured hR2D i values. The
alone and the complex between the full length of N and PTAIL [106]. In the NMR structure
blue arrows indicate the kinetic nature of changes in R2D .

9. Outlook
HS-AFM can visualize IDPs at the single molecule level and identify the structuralstates of their IDRs (e.g., fully disordered, partially folded, and fully folded states) andtheir dynamic transitions quantitatively. Importantly, the number of amino acids containedin a fully disordered IDR can be approximately estimated. In contrast, the ensemble NMR
spectroscopy methods are good at the atomistic determination of local structures andinteraction sites in IDPs but poor at detecting large-scale structures and identifying loosely
folded regions. Thus, HS-AFM and NMR mutually complement each other. As such, thecombination of these techniques can enrich our understanding of the dynamic structureof IDPs, as demonstrated in the studies of measles viral P protein. This review mainly
highlighted the structure and dynamics of IDPs revealed by HS-AFM and their functional
implications. However, a few HS-AFM studies have recently been carried out to observe
the functional phenomena of IDPs, such as amyloid fibril formation and dissolution andthe formation of liquid droplet-like condensates via LLPS. This new trend in HS-AFM
applications will be expanded in the near future because tools to analyze the functional
phenomena of IDPs are limited, while IDP-involved biological phenomena and diseases
are emerging in succession. In this regard, the HS-AFM studies on measles virus IDPs also
should be expanded to visualize dynamic processes, including N-P interactions, polymerization of N into NNUC , and even RNA synthesis. This line of studies may contribute to
the development of new drugs against measles; although the measles vaccine is alreadyavailable, many people still suffer and die from measles.
Funding: This research received no external funding.
Acknowledgments: The author acknowledges the wonderful collaboration with Sonia Longhi on
the HS-AFM studies of measles virus IDPs.

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Conflicts of Interest: The author declares no conflict of interest.

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