LiteFold/DisProt huggingface dataset:
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
Both populations of hNL3-cyt have high f/fo values, indicating that both are extended in solution.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Abnormal migration of the full-length hNL3-cyt domain on SDS-PAGE, as well as CD analysis of the heterogeneous hNL3-cyt solution in the far UV (Fig. S3 in Data S1), both provided early indications that hNL3-cyt is an IUP.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
To confirm this susceptibility to proteolytic digestion, the rate of digestion of hNL3-cyt by Proteinase K was compared to that of TcAChE, a globular protein that is known to be resistant to proteases in its native state (69) (Fig. 4). hNL3-cyt is, indeed, much more sensitive to Proteinase K than native TcAChE.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Evidence shows this peptide is disordered as shown in Table 1.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR spectra recorded for hNL3-cyt are typical of an unfolded protein: The signals are narrow and display very limited dispersion in the proton dimension. As can be seen in Fig. 12 A, the backbone amide proton chemical shifts are restricted to the coil region, i.e., 7.7–8.7 ppm.
The signal attenuation by dipolar cross relaxation (NOE) is so strong that it leads to negative crosspeaks. Such behavior is typical of unfolded proteins (75), whereas attenuation factors of 15% would be expected for the ordered regions in a folded protein with a molecular weight of 15 kDa.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: pre-molten globule (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Under denaturing conditions, in 8 M urea/100 mM NaH2PO4/10 mM Tris, pH 7.0, the 15 kDa protein eluted at a position corresponding to that to be expected for a ∼80 kDa globular protein, and to a hydrodynamic radius (RH) of ∼39 Å. In the absence of urea it eluted later, at a position corresponding to that for a ∼41 kDa globular protein, and to an RH of ∼28.4 Å, a radius that corresponds to a premolten globule according to the analysis of Uversky (23).
These data show that hNL3-cyt is already substantially unfolded under nondenaturing conditions, but becomes even more extended upon denaturation.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: fluorescence correlation spectroscopy evidence
Supporting statements:
Hydrodynamic radii of the nondenatured and denatured forms of hNL3-cyt, calculated using the Stokes-Einstein equation, were 25.0 ± 0.6 Å and 33.0 ± 0.8 Å, respectively. These values once again demonstrate that hNL3-cyt is disordered in solution, but can be further extended by denaturation.
Protein: DP00553 (UniProt Q9NZ94)
Region: residues 731	to 848
Annotation: disorder (Structural state)
Evidence: site-directed spin-labelling electron paramagnetic resonance evidence used in manual assertion
Supporting statements:
The shape of the ESR spectrum provides a measure of the mobility of the bound nitroxyl radical, which depends on the structure and flexibility of the protein in the vicinity of its site of attachment. The ESR signal of spin-labeled hNL3-cyt in 150 mM NaCl/10 mM sodium phosphate, pH 7.0, at 20°C reveals relatively sharp peaks (Fig. 11 A), indicating only limited restriction of the probe's movement.
Comparison of these values to that for isotropic rotation of the free radical in solution, τR ∼ 10−11−10−10 s, and to values for folded proteins, τR > 10−7 s, indicates limited restriction of the motion of the bound radical in buffer, and more substantial restriction in TFE.
Protein: DP00555 (UniProt Q16143)
Region: residues 1	to 134
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The measured values of the hydrodynamic radius for βS in its native state (32.4 Å) and in the presence of 8 M urea (35.4 Å) are both significantly shifted toward the value empirically calculated for a highly denatured state (36.0 Å) (Figure 1(b)).
The resonances are well resolved and sharp, with a limited dispersion of chemical shifts, reflecting the unfolded nature and high degree of backbone mobility.
Cross-references: BMRB:15298
Protein: DP00562 (UniProt Q9Y296)
Region: residues 11	to 25
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
flexible regions 11-25
Cross-references: PDB:2JSN
Protein: DP00570 (UniProt P09132)
Region: residues 1	to 144
Annotation: disorder (Structural state)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
Free SRP19 is a natively unstructured protein.
Protein: DP00570 (UniProt P09132)
Region: residues 1	to 144
Annotation: RNA binding (Molecular function)
Evidence: fluorescence resonance energy transfer evidence used in manual assertion
Supporting statements:
Thus, RNA binding and subsequent conformational changes in SRP19 were detected both as the initial change in the resonance energy transfer efficiency to the RNA-tethered Alexa 555 acceptor fluorophore and also as the local change in donor fluorophore environment (Figure 3A).
Protein: DP00573 (UniProt P03305)
Region: residues 202	to 215
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB shows missing electron densties in this region.
Cross-references: PDB:1QQP
Protein: DP00573 (UniProt P03305)
Region: residues 241	to 265
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The PDB shows missing electron densties in this region.
Cross-references: PDB:1QQP
Protein: DP00576 (UniProt P54252)
Region: residues 182	to 291
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Thus, we employed CD spectroscopy to estimate the secondary structure
content of the AT3182–291 variant (Fig. 2A). Spectra deconvolution
highlighted a highly disordered structure (63.7% random) with 22.4%
of α-helices
Protein: DP00576 (UniProt P54252)
Region: residues 182	to 291
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Two peaks for AT3182–291 were identified with Rg values of 1.95 ± 0.074 nm (Stoke radius 2.44 ± 0.095 nm) and 2.26 ± 0.003 nm (Stokes radius 2.84 ± 0.004 nm), respectively.
Protein: DP00576 (UniProt P54252)
Region: residues 194	to 361
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
the NMR spectrum of UIM_123 is suggestive of α-helical content with a large amount of disordered structure from the regions adjoining the UIM regions. This is in agreement with chemical shift index analysis of the NMR assignment data that showed α-helical stretches for UIM1 and -2 but was inconclusive for UIM3.
Protein: DP00576 (UniProt P54252)
Region: residues 224	to 348
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Overall, the C-terminal
tail is mostly unstructured but with regions of strong
helical propensity throughout, with maximal values in the
three UIMs (residues 224–240, 244-263, and 331–348), in
agreement with reports on peptides spanning the isolated
UIMs (17). Interestingly, also the residues in the polyQ tract
that could be assigned (298–305) exhibit some helical propensity.
Protein: DP00581 (UniProt P11233)
Region: residues 187	to 203
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
It has also been suggested that there is a second CaM-binding site within the disordered N-terminal extension of RalA (20). To unambiguously define the CaM-binding site on RalA, we carried out NMR-based chemical shift perturbation (CSP) experiments (Fig. 1A and SI Appendix, Fig. S1A) in the presence of Ca2+, utilizing 1H-15N heteronuclear single quantum correlation (HSQC) experiments recorded on 15N-labeled RalA· GMPPNP missing the last three residues (CΔ3, reference SI Appendix, Table S1).
Uniformly 13C,15N-labeled, HVR peptide was expressed in Escherichia coli and lipidated with MFn. NMR titrations of unlabeled CaM into the labeled peptide showed that residues within the core 10-residue motif were most affected (SI Appendix, Fig. S6).
This structure shows that the RalA-HVR forms only a single helical turn, remaining mainly disordered even when in complex with CaM.
The partially disordered HVR peptide in this complex, combined with flexibility in the orientations of the N- and C-lobes, is an unusual mode of CaM interaction, and there are only two other CaM-complex structures with this conformational flexibility
Cross-references: BMRB:34608
PDB:7NQC
Protein: DP00581 (UniProt P11233)
Region: residues 194	to 203
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
It has also been suggested that there is a second CaM-binding site within the disordered N-terminal extension of RalA (20). To unambiguously define the CaM-binding site on RalA, we carried out NMR-based chemical shift perturbation (CSP) experiments (Fig. 1A and SI Appendix, Fig. S1A) in the presence of Ca2+, utilizing 1H-15N heteronuclear single quantum correlation (HSQC) experiments recorded on 15N-labeled RalA· GMPPNP missing the last three residues (CΔ3, reference SI Appendix, Table S1).
The C-terminal Cys203 shifted significantly (CSP 0.19 ppm), but its position could also be tracked during the titrations (SI Appendix, Fig. S1A). In contrast, resonances corresponding to residues 194 to 202 of the HVR (SLAKRIRER) disappeared during the titration and gradually reappeared at different posi- tions as the titration progressed. This is known as slow exchange and indicates that the chemical shift differences between the free and CaM-bound states for these resonances are larger than the rate of exchange between these states. Such behavior demonstrates that these residues are involved in binding CaM, and the slow exchange rate between free and bound states strongly suggests that it is a high affinity interaction.
Overall, this analysis indicates that there is only one CaM-binding site in RalA, the core motif of which corresponds to 10 residues at the C terminus of the HVR.
Uniformly 13C,15N-labeled, HVR peptide was expressed in Escherichia coli and lipidated with MFn. NMR titrations of unlabeled CaM into the labeled peptide showed that residues within the core 10-residue motif were most affected (SI Appendix, Fig. S6).
Cross-references: BMRB:34608
PDB:7NQC
Protein: DP00581 (UniProt P11233)
Region: residues 194	to 203
Annotation: protein binding (Molecular function)
Evidence: radioisotope assay evidence used in manual assertion
Supporting statements:
The role of the C terminus of RalA in CaM binding was confirmed using scintillation proximity assays (SPA), where [3H] GTP·RalA was titrated into His-tagged CaM immobilized on an SPA bead (SI Appendix, Fig. S1B).
Protein: DP00581 (UniProt P11233)
Region: residues 194	to 203
Annotation: protein binding (Molecular function)
Evidence: isothermal titration calorimetry evidence used in manual assertion
Supporting statements:
We synthesized two maleimide-functionalized prenyl anchor mimics (SI Appendix, Fig. S2A), MFn (maleimido-farnesyl), which includes a 15-carbon far- nesyl group, and MGG (maleimido-geranylgeranyl), which includes a 20-carbon geranylgeranyl. RalA CΔ3 prenylated with MGG was insoluble in the absence of detergents but could be stabilized by binding to nanodisc membrane mimics. Titration of CaM into nanodisc-bound RalA CΔ3 MGG resulted in a return of heat changes and an apparent binding affinity of 3.8 μM (Fig. 1B).
Protein: DP00581 (UniProt P11233)
Region: residues 194	to 203
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
CaM was immobilized on the surface of a CM5 sensor chip via amine coupling and multicycle experiments recorded by flowing either RalA CΔ3 or RalA CΔ3-MFn over the immobilized protein (Fig. 1C). Dissociation constants for unmodified and prenylated RalA calculated by 1:1 equilibrium analysis (Fig. 1D) were 214 ± 27 nM and 19 ± 3 nM, respectively.
Protein: DP00581 (UniProt P11233)
Region: residues 1	to 10
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Even though the N terminus was present in the constructs of Ral used for the present study, the first ten residues are disordered and are not observed in the electron density maps for any of the structures.
Cross-references: PDB:1UAD
Protein: DP00583 (UniProt P16006)
Region: residues 63	to 82
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The final model (Figure 3a) includes residues 1−62 and 84−193 of the 193-residue molecule. The absence of electron density for residues 63−83 indicates that these residues form a highly flexible loop that is disordered in the crystal.
Cross-references: PDB:1VQ2
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
It is a well-known fact that binding Coomassie Brilliant Blue R-250 dye depends on a positively charged residue with adjoining hydrophobic residues.(20) This was probably the reason why Stm, a protein of extremely low hydrophobicity, exhibited lowered Commassie-binding.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectrum of StmΔSP is typical for a disordered protein, as can be seen from its large negative ellipticity at 198 nm (Figure 5B).
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
According to published data, IDPs exhibit a substantially increased hydrodynamic volume relative to globular proteins, leading to a significant increase in their apparent molecular mass.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder (Structural state)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
The emission spectrum has a maximum fluorescence (λmax) of 350 nm, suggesting the occurrence of the W residue in a polar environment. This suggests that it is unlikely that Stm possesses an extensive hydrophobic core, otherwise, the W residue would probably be engaged in its formation.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: phosphorylation display site (Disorder function)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
We observed different levels of Stm phosphorylation depending on the ATP concentration in the solution. The largest shift in Stm mobility was observed when ATP concentrations in the reaction mixture were 0.5 and 5 mM.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: calcium ion binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
We determined that Stm is a calcium binding protein by using a quantitative calcium affinity assay(32) based on the competition between Stm and the fluorescent probe sensitive to calcium ions (Rhod-5N) (Figure 2).
Calcium binding by Stm and StmP was observed as a Rhod-5N fluorescence signal in samples containing Stm and StmP lower than that in the control samples (Figure 2). Fluorescence intensities were used to calculate the affinity of Stm and StmP for Ca2+. The results are summarized in Table 2.
The results of the calcium binding assay showed that Stm bound 28 calcium ions per molecule with an apparent KD of 210 μM.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder to pre-molten globule (Structural transition)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
Calcium ions caused a decrease in the hydrodynamic radius of both StmP and Stm (Figure 3).
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder to pre-molten globule (Structural transition)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
In contrast to gel filtration, sedimentation velocity reveals that structures of Stm and StmP are different, as suggested by the difference in the frictional ratio. 
These results confirm that the decrease in the hydrodynamic radius of Stm and StmP in the presence of calcium is caused by compaction of the molecule rather than by dissociation of the subunits.
Protein: DP00584 (UniProt A2VD23)
Region: residues 1	to 613
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of Stm and StmP exhibited shapes typical for IDPs. There was a large minimum near 200 nm, which is characteristic for unfolded proteins.
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
A trypsin digestion test indicates that C82 is significantly sensitive to degradation (Fig. 2C)
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The C82 CD spectrum shows a minimum near 200 nm indicating that this protein is a random coil that lacks apparent secondary structure (Fig. 3A).
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Although there is a poor 1HN chemical shift distribution in the HSQC of C82 (0.95 ppm), which is typical of proteins that possess little tertiary structure and are disordered, we observed 63 of the 69 expected peaks. Moreover, the NOESY spectra of C82 contain few NOE correlations which is indicative of the absence of secondary structure.
As shown in Figure 4D, CSI Cα of C82 is oscillating around zero, suggesting that C82 exists in an unstructured conformation.
Cross-references: BMRB:15768
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The NOEs, with an average of 0.51, are significantly higher than those observed for C82 in water (Duvignaud et al. 2009) (Fig. 4) and indicate that C82 gains significant order in TFE, although probably not as ordered as a typical globular protein (Fig. 4).
In aqueous solution this region is disordered, as shown by NOEs typical of proteins without any stable secondary structure (Fig. 4).
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Authors show the region is intrinsically unstructured by its large negative ellipticity at 200 nm in a circular dichroism study (Fig. 1E).
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: viral capsid assembly (Biological process)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
To confirm our final product, we performed an SDS–PAGE electrophoresis (Fig. 2A) and an in vitro assembly assay showing that the C82 was able to assemble into NLPs in presence of tRNA as seen by electron microscopy (Fig. 2B).
However, C82 is able to organize itself in vitro into NLPs in the presence of structured RNA.
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: RNA binding (Molecular function)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
To confirm our final product, we performed an SDS–PAGE electrophoresis (Fig. 2A) and an in vitro assembly assay showing that the C82 was able to assemble into NLPs in presence of tRNA as seen by electron microscopy (Fig. 2B).
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: protein binding (Molecular function)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
To confirm our final product, we performed an SDS–PAGE electrophoresis (Fig. 2A) and an in vitro assembly assay showing that the C82 was able to assemble into NLPs in presence of tRNA as seen by electron microscopy (Fig. 2B).
However, C82 is able to organize itself in vitro into NLPs in the presence of structured RNA.
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 82
Annotation: disorder (Structural state)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
It should be noted that C82 migrates at an abnormal molecular weight of ∼15 kDa instead of the expected ∼10 kDa.
Protein: DP00588 (UniProt P27958)
Region: residues 1	to 79
Annotation: viral capsid assembly (Biological process)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
We showed that the N-terminal 75 aa (C 1–75) were sufficient to trigger the formation of NLPs in vitro, while the construct C 1–71 failed. The C 1–75 protein contains only four additional amino acids (PEGR), which delimit the border of the second basic cluster of the C protein, which can play an important role in virus assembly. More than 70 % of the structures observed with the C 1–75 protein were, however, aggregates with a diameter exceeding the expected range for the HCV nucleocapsid. The addition of a further four amino acids in the construct C 1–79 stabilized the particles and gave rise to uniform and structured particles resembling HCV nucleocapsids. Also, the NLPs produced from C 1–82 formed one band with a density of 1·32 g ml−1 in a CsCl gradient, suggesting that all the NLPs were uniform in diameter, appearance and density (Fig. 5F5). Based on these results, we suggest that the minimal assembly domain of the HCV C protein resides in the first 79 aa of the protein.