LiteFold/DisProt huggingface dataset:
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 139
Annotation: vesicle docking (Biological process)
Evidence: bait-prey hybrid interaction evidence used in manual assertion
Supporting statements:
We found that the cross-linked Syt1 mutants could reduce vesicle docking by approximately 40%. Although the changes appear to be statistically insignificant, the inhibition of vesicle docking could be recovered when the sample was treated with DTT within experimental errors (Figure 4B). Thus the results suggest that the flexibility of the linker region is favourable for Syt1 in assisting vesicle docking.
Thus the results of the present study support the notion that an extended and flexible linker region is preferred for Syt1 to induce vesicle docking to the plasma membrane, whereas a shorter linker region is favoured for Syt1 to drive fusion pore opening.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 139
Annotation: vesicle fusion (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
In contrast, in the content-mixing assay, cross-linked Syt1 enhanced fusion pore opening compared with wild-type Syt1 by approximately 50% (Figures 4C and 4D), which also could be negated by DTT. Thus the results show that the folded linker region is preferable for Syt1/Ca2+ to drive opening of the fusion pore for content mixing.
Thus the results of the present study support the notion that an extended and flexible linker region is preferred for Syt1 to induce vesicle docking to the plasma membrane, whereas a shorter linker region is favoured for Syt1 to drive fusion pore opening.
Protein: DP04144 (UniProt P21707)
Region: residues 99	to 140
Annotation: vesicle docking (Biological process)
Evidence: loss-of-function mutant phenotype evidence used in manual assertion
Supporting statements:
To further substantiate this observation, we made the linker of Syt1 shorter by deleting 40 amino acids [Δ(99–140)aa]. As expected, the truncated mutant of Syt1 reduced the vesicle docking by ~50%, but enhanced fusion pore opening by more than 40% (Figure 4E). Thus the results of the present study support the notion that an extended and flexible linker region is preferred for Syt1 to induce vesicle docking to the plasma membrane, whereas a shorter linker region is favoured for Syt1 to drive fusion pore opening.
Protein: DP04144 (UniProt P21707)
Region: residues 99	to 140
Annotation: vesicle fusion (Biological process)
Evidence: gain-of-function mutant phenotypic evidence used in manual assertion
Supporting statements:
To further substantiate this observation, we made the linker of Syt1 shorter by deleting 40 amino acids [Δ(99–140)aa]. As expected, the truncated mutant of Syt1 reduced the vesicle docking by ~50%, but enhanced fusion pore opening by more than 40% (Figure 4E). Thus the results of the present study support the notion that an extended and flexible linker region is preferred for Syt1 to induce vesicle docking to the plasma membrane, whereas a shorter linker region is favoured for Syt1 to drive fusion pore opening.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 139
Annotation: calcium ion sensor activity (Molecular function)
Evidence: site-directed spin-labelling electron paramagnetic resonance evidence used in manual assertion
Supporting statements:
We found that the linker was in a flexible conformation when reconstituted on v-vesicles (Figure 5A, □). Upon binding to t-vesicles in the absence of Ca2+, the linker region became slightly more flexible as the EPR lines got somewhat narrower (Figure 5A, ○). However, an opposite conformation change happened when Syt1 bound to t-vesicles with Ca2+ (Figure 5A, △), which indicated a conformation change from flexible to restricted.
Protein: DP04144 (UniProt P21707)
Region: residues 80	to 139
Annotation: flexible linker (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
The 60-residue-long linker can theoretically extend as much as 30 nm, which will allow the C2AB domain to reach out to bind t-SNARE on the target membrane [16,17].
To further substantiate this observation, we made the linker of Syt1 shorter by deleting 40 amino acids [Δ(99–140)aa]. As expected, the truncated mutant of Syt1 reduced the vesicle docking by ~50%, but enhanced fusion pore opening by more than 40% (Figure 4E). Thus the results of the present study support the notion that an extended and flexible linker region is preferred for Syt1 to induce vesicle docking to the plasma membrane, whereas a shorter linker region is favoured for Syt1 to drive fusion pore opening.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Regardless, we were still able to assess residual secondary structure from the residues examined by subtracting IDP/IDR-specific random coil chemical shifts from the observed Cα, Cβ, and carbonyl carbon chemical shifts. (25) In a majority of assigned residues subjected to this secondary structure analysis, there appears to be no strong preference for residual structure; both Cα–Cβ and carbonyl carbon chemical shift differences alternate between positive and negative values, indicating this linker region is largely random coil in solution (Figure 3B,C).
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: flexible linker (Disorder function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
Regardless, we were still able to assess residual secondary structure from the residues examined by subtracting IDP/IDR-specific random coil chemical shifts from the observed Cα, Cβ, and carbonyl carbon chemical shifts. (25) In a majority of assigned residues subjected to this secondary structure analysis, there appears to be no strong preference for residual structure; both Cα–Cβ and carbonyl carbon chemical shift differences alternate between positive and negative values, indicating this linker region is largely random coil in solution (Figure 3B,C).
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: flexible linker (Disorder function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
This finding is consistent with circular dichroism measurements of the IDR (Figure 3D) and predictions of Composition Profiler (Table S2). However, there may still be minor residual helical content given that equilibration with increasing concentrations of urea results in the absorption profile of the juxtamembrane linker more closely resembling that of random coil (Figure 3D).
For DSC and CD experiments for which small quantities of protein are needed, the linker region peptide specifically encoding residues 81–157 was produced via solid state synthesis through the University of Minnesota Genomics Center: KKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNN. Inclusion of residues 143–157, which corresponds to part of C2A’s first β-strand, was done so that the peptide would contain absorbing residues for measuring concentration.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
This finding is consistent with circular dichroism measurements of the IDR (Figure 3D) and predictions of Composition Profiler (Table S2). However, there may still be minor residual helical content given that equilibration with increasing concentrations of urea results in the absorption profile of the juxtamembrane linker more closely resembling that of random coil (Figure 3D).
For DSC and CD experiments for which small quantities of protein are needed, the linker region peptide specifically encoding residues 81–157 was produced via solid state synthesis through the University of Minnesota Genomics Center: KKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNN. Inclusion of residues 143–157, which corresponds to part of C2A’s first β-strand, was done so that the peptide would contain absorbing residues for measuring concentration.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: lipid binding (Molecular function)
Evidence: differential scanning calorimetry evidence used in manual assertion
Supporting statements:
These results suggest that, with a synaptic vesicle-like environment, Syt I’s juxtamembrane IDR can exist in a membrane-associated state that has measurable heat capacity. While DSC does not provide direct information about the type of folded structure, the small endotherm (particularly on the reversibility scan, dashed light green) does indicate weak intramolecular interactions within the IDR when it is membrane-associated.
For DSC and CD experiments for which small quantities of protein are needed, the linker region peptide specifically encoding residues 81–157 was produced via solid state synthesis through the University of Minnesota Genomics Center: KKCLFKKKNKKKGKEKGGKNAINMKDVKDLGKTMKDQALKDDDAETGLTDGEEKEEPKEEEKLGKLQYSLDYDFQNN. Inclusion of residues 143–157, which corresponds to part of C2A’s first β-strand, was done so that the peptide would contain absorbing residues for measuring concentration.
In the presence of a complex lipid mixture that mimics the outer leaflet of a synaptic vesicle (Figure 1B), we found through DSC (Figure 2) and NMR (Figure 4) measurements that the IDR experiences endotherm and chemical shift changes, respectively, consistent with IDR–synaptic lipid interactions, though in a mostly disordered structural state.
Synaptic vesicle mimic were prepared in a final cholesterol content of ∼31% of total lipid and with the lipids: 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0, 22:6 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0, 18:1 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0, 18:1 PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoserine (18:0, 22:6 PS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (18:0, 18:1 PS), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho(1′-myo-inositol-4′,5′-bisphosphate) [18:0, 20:4 PI(4,5)P2], 1,2-dioleoyl-sn-glycero-3-phospho(1′-myo-inositol-4′,5′-bisphosphate) [18:1, 18:1 PI(4,5)P2], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (16:0, 18:1 PI).
Protein: DP04145 (UniProt P21579)
Region: residues 101	to 119
Annotation: lipid binding (Molecular function)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
POPC:POPS SUVs showed little to no effect, largely causing peak broadening (and disappearance) presumably because of membrane association (Figure 4A). In contrast, synaptic lipid SUVs caused more pronounced spectral changes. For example, residues A101, I102, V107, T113, A118, and L119 seem to experience larger changes in their local environment as their membrane-free chemical shifts are missing in the presence of synaptic lipid SUVs (Figure 4B–D). More generally, the spectra show greater peak dispersion and some chemical exchange (Figure 4D).
In the presence of a complex lipid mixture that mimics the outer leaflet of a synaptic vesicle (Figure 1B), we found through DSC (Figure 2) and NMR (Figure 4) measurements that the IDR experiences endotherm and chemical shift changes, respectively, consistent with IDR–synaptic lipid interactions, though in a mostly disordered structural state.
Synaptic vesicle mimic were prepared in a final cholesterol content of ∼31% of total lipid and with the lipids: 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0, 22:6 PE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (18:0, 18:1 PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0, 18:1 PE), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoserine (18:0, 22:6 PS), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (18:0, 18:1 PS), 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho(1′-myo-inositol-4′,5′-bisphosphate) [18:0, 20:4 PI(4,5)P2], 1,2-dioleoyl-sn-glycero-3-phospho(1′-myo-inositol-4′,5′-bisphosphate) [18:1, 18:1 PI(4,5)P2], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (16:0, 18:1 PI).
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: vesicle organization (Biological process)
Evidence: carboxyfluorescein diacetate succinimidyl ester staining evidence used in manual assertion
Supporting statements:
When the CF-containing vesicles were cooled through their transition temperature in the presence of juxtamembrane linker peptide, the percentage of maximal dye efflux increased by 28 relative to that of the liposomes alone (Figure 7). As a positive control, we also performed the experiment with Ca2+-bound short C2A and found that the percentage of maximal dye efflux was increased by 15. This indicates that the magnitude of dye efflux with the IDR is likely relevant to destabilizing the membrane as maximal efflux exceeds that of the known C2A intercalator. Consistent with the EPR measurements of partial insertion, the juxtamembrane linker destabilizes the membrane. This result may be relevant to protein intercalation as a way of destabilizing the membrane and consequently lowering the energetic barrier to fusion.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 142
Annotation: regulation of calcium ion binding (Biological process)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
In this case, the σ of ∼4 indicates that residues 96–139 confer modest positive cooperativity to C2A’s Ca2+-binding sites, consistent with terbium binding studies performed previously on these same two C2A constructs.
The end result of IDR inclusion, however, appears to be pronounced alteration of the thermodynamic parameters describing Ca2+ binding. Long C2A is still Ca2+ binding-competent as evidenced by the fact that addition of Ca2+ to the long C2A construct in the absence of any lipid ligand still results in an elevated unfolding temperature (Figure 6C) as would be expected from chelation of ligand.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 98
Annotation: presynaptic membrane (Cellular component)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Out of line with these results, unexpectedly, there is a comparatively lower staining by the h-FJMS peptide of the SVs, which are largely localized inside the motor axon terminal, as documented by comparison with the staining of an antibody specific for the vesicular acetylcholine transporter (Fig. 6). Taken together these data suggest that a large part of the fluorescent peptide inserts into the presynaptic membrane.
Protein: DP04145 (UniProt P21579)
Region: residues 81	to 98
Annotation: phospholipid binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Fig. 7 shows that the h-FJMS indeed binds anionic phospholipids, including phosphatidylserine and phosphatidylinositides (PIs), which are enriched in the SV membrane and on the cytosolic face of the PM [39], [41].
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: disorder (Structural state)
Evidence: synchrotron radiation circular dichroism evidence used in manual assertion
Supporting statements:
Spectral deconvolution of N-terminal domain spectrum using Bestsel
software [46] suggests the high disordered content of the N-terminal
domain (51.5% unordered structures, 2.5% α-helix, 27.3% β-strand,
18.7% turn).
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: order (Structural state)
Evidence: synchrotron radiation circular dichroism evidence used in manual assertion
Supporting statements:
N-terminal domain tends to lose disordered structures in favor of ß-strands (from 51.5% unordered structures, 27.3% ß-strands at 55% ethanol (v/v) to 41.1% unordered structures, 38.9% ß-strands at 20% ethanol (v/v)).
The repetitive N-terminal domain appears to fold into ß-strand structures while the non-repetitive C-terminal domain seems to gain α-helix and disordered content in detriment to the β structures. The presence of disorder to order transitions within the N-terminal may be consistent with the propensity of IDPs to this type of
transition, due their flexibility and lack of structural constraint
[25,47,48].
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: disorder to order (Structural transition)
Evidence: synchrotron radiation circular dichroism evidence used in manual assertion
Supporting statements:
N-terminal domain tends to lose disordered structures in favor of ß-strands (from 51.5% unordered structures, 27.3% ß-strands at 55% ethanol (v/v) to 41.1% unordered structures, 38.9% ß-strands at 20% ethanol (v/v)).
The repetitive N-terminal domain appears to fold into ß-strand structures while the non-repetitive C-terminal domain seems to gain α-helix and disordered content in detriment to the β structures. The presence of disorder to order transitions within the N-terminal may be consistent with the propensity of IDPs to this type of transition, due their flexibility and lack of structural constraint [25,47,48].
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Contrary to the C-terminal domain, N-terminal domain exhibit over-estimated
apparent molecular weight (SDS-PAGE, Fig. 2A&B). This electrophoretic profile was also observed in the case of the γ44-gliadin full length [36] and suggests a conformational specificities of the domain, in line with our previous predictions. Since disordered proteins are known for their abnormal electrophoretic mobility due to their hydrodynamic radius, higher than those of the globular protein (such as C-terminal domain) [44,45], this may suggest that the repetitive N-terminal is indeed disordered.
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
The N-terminal domain radius is equivalent to the whole protein radius. These results may emphasize the expanded conformation of the N-terminal domain contrasting with the compact conformation of the C-terminal domain. As aforementioned, disordered proteins have larger hydrodynamic dimensions compared to conventional globular proteins [33,44].
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 162
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
The disorder of the N-terminal domain and the order of the C-terminal domain are visible in the shape of the pair distance. distribution functions p(r) (Fig. 6B). The maximum distance (Dmax) found for γ44-gliadin, N-terminal domain and C-terminal domain are respectively of 255 Å, 150 Å and 100 Å. The large Dmax of the N-terminal domain suggests an extended conformation, while the lower Dmax of the C-terminal domain suggests a compact conformation.
Finally, SAXS experiments provide ensemble-averaged structural information about the γ44-gliadin and its isolated domains. All structural data obtained are summarized in Table 3. SAXS experiments are complementary to CD results. They also suggest that the N-terminal domain is unordered in contrast to the globular-like shape of the C-terminal domain.
Protein: DP04148 (UniProt P08453)
Region: residues 1	to 327
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
Microscopic observations reveal that this increase of absorbance values is due to highly dynamic and spherical micrometric droplets (Fig. 4).
The number and the size of formed droplets increase with increasing protein concentration (Fig. 4B).
Microscopic imaging shows a decrease of the number of droplets with decreasing protein concentration until a total disappearance at low protein concentration (Fig. 4C). These observations show the ability of γ44-gliadin to form reversible assemblies upon solvent quality change.
Protein: DP04149 (UniProt P51788)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The refined atomic model contains residue numbers from 89 to 560, fitting well within the density of our TMD map, but lacking the flexible N- and C-terminal regions (Fig. 1b).
Cross-references: PDB:7XF5
EMDB:EMD-33169
Protein: DP04149 (UniProt P51788)
Region: residues 650	to 753
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The refined atomic model contains residue numbers from 89 to 560, fitting well within the density of our TMD map, but lacking the flexible N- and C-terminal regions (Fig. 1b).
However, the cytosolic C-terminal domain (CTD) had lower resolution, suggesting that the two tandem-linked CBS domains are quite flexible.
Cross-references: PDB:7XF5
EMDB:EMD-33169
Protein: DP04149 (UniProt P51788)
Region: residues 846	to 898
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The refined atomic model contains residue numbers from 89 to 560, fitting well within the density of our TMD map, but lacking the flexible N- and C-terminal regions (Fig. 1b).
However, the cytosolic C-terminal domain (CTD) had lower resolution, suggesting that the two tandem-linked CBS domains are quite flexible.
Cross-references: PDB:7XF5
EMDB:EMD-33169
Protein: DP04149 (UniProt P51788)
Region: residues 561	to 572
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The cryo-EM structure of the full length apo protein shows this region lacks electron density, indicating it is disordered. 
Cross-references: PDB:7XF5
EMDB:EMD-33169
Protein: DP04149 (UniProt P51788)
Region: residues 645	to 743
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
In both classes, the CBS domains can be fit to the density; however, residues 645–743 in the CLC-2 CTD are not resolved and thus are omitted from the models and from the Q-score calculation (Pintilie et al., 2020, Figure 4—figure supplement 1).
Cross-references: PDB:8TA4
PDB:8TA5
EMDB:EMD-41129
EMDB:EMD-41128