LiteFold/DisProt huggingface dataset:
Protein: DP00223 (UniProt P14635)
Region: residues 1	to 78
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of ND-cycB and ND-Pds1 (Fig. 3) are devoid of the significant signatures of secondary structure, most notably negative bands at 222 nm and 208 nm for α-helix and a negative band at 217 nm for β-sheet. Instead, the predominant feature is a strong negative band at 200 nm, which is correlated with a predominantly unstructured polypeptide.
A more compelling analysis involves examination of the degree of cooperativity in the change of CD spectra with increasing temperature. Cooperativity seems to be an unfailing hallmark of the folded-to-unfolded transition. As can be seen from measurements at the two characteristic wavelengths of 200 nm and 222 nm (Fig. 3), the variation of the ellipticity values with temperature does not display cooperativity; instead, the values vary linearly.
Protein: DP00225 (UniProt Q05140)
Region: residues 745	to 896
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The first fragment extended from Val328to Gly745, and the second from Gly745 to Leu896. As expected, the spectra of both fragments revealed little secondary structure; but more important, the sum of the molar ellipticities in the spectra of the three recombinant AP180 fragments (His6-AP180-(1–329), -(328–745), and -(745–896)) was identical to that in the spectrum of recombinant full-length AP180 and similar to that in the spectrum of AP180 isolated from pig brain (Fig.4 D).
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In the spectra of the fragments lacking the ENTH domains (His6-epsin 1-(144–575) and His6-AP180-(328–896)), the α-helical characteristics were almost completely lost, whereas the content of random structures increased from 56 to 66% for the epsin 1 fragment and from 55 to 84% for the AP180 fragment (Fig. 4,A and B).
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 745
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The first fragment extended from Val328to Gly745, and the second from Gly745 to Leu896. As expected, the spectra of both fragments revealed little secondary structure; but more important, the sum of the molar ellipticities in the spectra of the three recombinant AP180 fragments (His6-AP180-(1–329), -(328–745), and -(745–896)) was identical to that in the spectrum of recombinant full-length AP180 and similar to that in the spectrum of AP180 isolated from pig brain (Fig.4 D).
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: disorder (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The observed hydrodynamic size is consistent with either an extended rod with a thickness of 1.5–2.0 nm and a length of some 50 nm or, alternatively, a poorly folded polypeptide chain. Taken together, our data obtained from quantitative gel filtration chromatography and ultracentrifugation strongly suggest that the carboxyl-terminal parts of epsin 1 and AP180 are rather extended, with the consequence that they behave like very large proteins on gel filtration columns and like molecular parachutes during ultracentrifugation.
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
The observed hydrodynamic size is consistent with either an extended rod with a thickness of 1.5–2.0 nm and a length of some 50 nm or, alternatively, a poorly folded polypeptide chain. Taken together, our data obtained from quantitative gel filtration chromatography and ultracentrifugation strongly suggest that the carboxyl-terminal parts of epsin 1 and AP180 are rather extended, with the consequence that they behave like very large proteins on gel filtration columns and like molecular parachutes during ultracentrifugation.
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: disorder (Structural state)
Evidence: protein thermal shift assay evidence used in manual assertion
Supporting statements:
SDS-PAGE analysis of the supernatant and pellet fractions indeed demonstrated that His6-epsin 1-(144–625) and His6-AP180-(328–896) were both heat-stable, whereas the full-length proteins and GST added as a carrier and internal standard did almost quantitatively precipitate (Fig.5 A).
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: clathrin binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
Binding of the boiled protein fragments to clathrin and the adaptor was assessed by pull-down experiments using the immobilized recombinant α-appendage domain and the clathrin amino-terminal domain as baits. No significant differences between the untreated and heat-treated His6-AP180-(328–896) fragments were observed (Fig.5 B).
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: clathrin coat assembly (Biological process)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
Similarly, heating did not affect the ability of the fragment to induce clathrin assembly (Fig. 5 C).
A, Coomassie Blue staining/SDS-PAGE of the supernatants (s) and pellets (p) after ultracentrifugation of the reaction mixtures showed that full-length epsin 1 very efficiently assembled clathrin. His6-epsin 1-(144–575) also assembled clathrin, albeit less efficiently than the full-length protein.
Protein: DP00225 (UniProt Q05140)
Region: residues 328	to 896
Annotation: clathrin coat assembly (Biological process)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
As expected, this epsin 1 fragment also possessed assembly activity, although it was somewhat lower compared with that of the full-length protein. Electron micrographs showed that the cages were slightly larger and less homogeneous than those assembled by AP180 or His6-AP180-(328–896) (Fig. 6).
Free clathrin triskelia were incubated with recombinant full-length epsin 1, a recombinantly expressed fragment of epsin 1 lacking the ENTH domain, and 6x-His-AP180-(328–896) as a positive control.
Protein: DP00226 (UniProt Q9CXW3)
Region: residues 48	to 77
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 30 C-terminal residues corresponding to SIP(48−77) did not form regular secondary structure.
Cross-references: PDB:1YSM
Protein: DP00226 (UniProt Q9CXW3)
Region: residues 48	to 77
Annotation: molecular adaptor activity (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
SIP appears not to engage in specific catalytic activity but is part of a growing family of adaptor proteins that mediate the correct assembly of ubiquitination complexes to achieve functional diversity.
The core of the complex is formed by a novel Cullin-1-like adaptor protein SIP, which provides a physical link between the RING domain of Siah-1 and the substrate engaged by the Skp1/Ebi F-Box protein (Figure 1).
Protein: DP00226 (UniProt Q9CXW3)
Region: residues 48	to 77
Annotation: ubiquitin protein ligase binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR titrations of unlabeled Siah(90−282) into 15N-labeled intact SIP resulted in a loss of peak intensity and chemical shift changes for residues (Met46−Gly71) in the linker and, surprisingly, the disappearance of residues from the CS domain (Figure 6A).
For the SIP N-terminal region (residues 1−77), differential line broadening, chemical shift perturbations, and a loss in peak intensity of residues located in the linker region are observed as in the intact protein (Figure 6B). In contrast, the NMR signals of the isolated CS domain behaved differently than those in the intact protein, as no perturbations in the CS domain were observed (Supporting Information).
Protein: DP00226 (UniProt Q9CXW3)
Region: residues 178	to 229
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Figure 2 shows the HSQC spectra for residues 1−77, 74−178, and 178−229. The dispersion of NMR resonances clearly shows that the N-terminal and central fragments contain independent folded globular domains, whereas the C-terminal fragment is not folded. Additional spectra were acquired for residues 155−229 and 73−229 (Supporting Information), but the lack of signal dispersion for the C-terminal residues was not affected by extending the N-terminus of these constructs.
Protein: DP00226 (UniProt Q9CXW3)
Region: residues 48	to 77
Annotation: flexible linker (Disorder function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The 30 C-terminal residues corresponding to SIP(48−77) did not form regular secondary structure.
Structural characterization of SIP(1−77) revealed the putative Siah-1 binding region (Pro61−Pro67, PAAVVAP) is part of the unstructured linker region (residues 48−73) that connects the N-terminal helical domain of SIP and the CS domain.
Protein: DP00228 (UniProt P07293)
Region: residues 724	to 760
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
Maxima and minima in the CD spectra, which indicate a helical structure for peptide A, were not seen for peptide C (Figure 7C). Thus peptide C has a random-coil structure and therefore adopts different conformations, each maintained briefly.
Protein: DP00228 (UniProt P07293)
Region: residues 724	to 760
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The simplicity of cross peaks in the amide-α region also indicates a lack of one predominant structure (Figure 7A).
Protein: DP00229 (UniProt P05455)
Region: residues 326	to 408
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
NMR analysis of this series of deletion mutants allowed us to identify a core globular domain, comprising residues 231–325 (Jacks et al. 2002; and see below), flanked by a short flexible N terminus (residues 225–230) and a long, flexible C terminus (residues 326–408).
In particular, comparison of 1H-15N HSQC spectra of La225–408, La225–359, and La225–334 (Figure 2) indicated that all well-dispersed peaks correspond to residues assigned in the 230–325 region (Jacks et al. 2002) and suggests, first, that the three-dimensional structure of this fragment is very similar in all three cases, regardless of the length of the C-terminal tail and, second, that the C-terminal tail (326–408) is itself unstructured.
Protein: DP00229 (UniProt P05455)
Region: residues 326	to 408
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Proteolysis during expression gave rise to a hexahistidine-tagged recombinant protein fragment (La225–334), which was purified by affinity chromatography in a single step on a Ni-NTA resin column (Qiagen; Jacks et al. 2002).
Protein: DP00229 (UniProt P05455)
Region: residues 335	to 408
Annotation: poly(U) RNA binding (Molecular function)
Evidence: electrophoretic mobility shift assay evidence used in manual assertion
Supporting statements:
Together, these data reveal that a region present in La225–359 and La225–408, but absent in La225–334, is capable of binding in a nonspecific fashion with 5′-monophosphorylated 10 nt oligo(U) RNA. The interacting portion therefore does not encompass the atypical C-terminal RRM (231–325), and probably involves the unstructured SBM that spans residues 328–363, an observation that is consistent with previous work on pre-tRNA (Fan et al. 1998), Hepatitis C virus IRES (Ali et al. 2000), and 5′-triphosphate RNA targets (Bhattacharya et al. 2002).
Protein: DP00233 (UniProt P14448)
Region: residues 19	to 44
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The amino-terminal segments of the α and β chains, including fibrinopeptides A and B, are not visible in electron density maps, however, and must be highly disordered. The αC domain is also very disordered.
Electron density maps barely reveal the outlines of the disordered carboxyl regions of the α chains (Figure 2), and the amino-terminal segments of the α and β chains, including fibrinopeptides A and B, could not be resolved. The reasonable R-factors found after refinement (Table 1) indicate that these parts of the molecule do not contribute significantly to the diffraction and must be highly disordered. That the missing portions had not been lost to degradation during incubations leading to crystallization was ascertained by SDS gels and amino-terminal sequencing.
Cross-references: PDB:1M1J
Protein: DP00233 (UniProt P14448)
Region: residues 239	to 505
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The αC domain is also very disordered.
The disordered αC domains reside in the sinuses of the coiled coils mostly away from the central domain (Figure 2).
Electron density maps barely reveal the outlines of the disordered carboxyl regions of the α chains (Figure 2), and the amino-terminal segments of the α and β chains, including fibrinopeptides A and B, could not be resolved. The reasonable R-factors found after refinement (Table 1) indicate that these parts of the molecule do not contribute significantly to the diffraction and must be highly disordered. That the missing portions had not been lost to degradation during incubations leading to crystallization was ascertained by SDS gels and amino-terminal sequencing.
Cross-references: PDB:1M1J
Protein: DP00236 (UniProt P02686)
Region: residues 219	to 229
Annotation: order to disorder (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The CSI analyses of our assignments, shown in Fig. 3, indicate a noticeable tendency of a central 10-residue segment of the polypeptide to adopt a helical conformation from residues 5′ to 14′, for samples in TFE-d2 (Fig. 3B) and in DPC-d38 (Fig. 3C), but not in KCl (Fig. 3A).
Cross-references: BMRB:6857
Protein: DP00236 (UniProt P02686)
Region: residues 215	to 231
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The CSI analyses of our assignments, shown in Fig. 3, indicate a noticeable tendency of a central 10-residue segment of the polypeptide to adopt a helical conformation from residues 5′ to 14′, for samples in TFE-d2 (Fig. 3B) and in DPC-d38 (Fig. 3C), but not in KCl (Fig. 3A).
Cross-references: BMRB:6857
Protein: DP00236 (UniProt P02686)
Region: residues 215	to 231
Annotation: order (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In aqueous solution (pure water, and 100 mm KCl, pH 6.5), the spectra indicated that the polypeptide had little or no regular secondary structure. In organic and membrane-mimetic conditions (30% TFE and 20 mm DPC, respectively), the spectra clearly indicated an α-helical conformation.
Protein: DP00236 (UniProt P02686)
Region: residues 215	to 231
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In aqueous solution (pure water, and 100 mm KCl, pH 6.5), the spectra indicated that the polypeptide had little or no regular secondary structure. In organic and membrane-mimetic conditions (30% TFE and 20 mm DPC, respectively), the spectra clearly indicated an α-helical conformation.
Protein: DP00236 (UniProt P02686)
Region: residues 215	to 231
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In aqueous solution (pure water, and 100 mm KCl, pH 6.5), the spectra indicated that the polypeptide had little or no regular secondary structure. In organic and membrane-mimetic conditions (30% TFE and 20 mm DPC, respectively), the spectra clearly indicated an α-helical conformation.
Protein: DP00236 (UniProt P02686)
Region: residues 219	to 229
Annotation: lipid binding (Molecular function)
Evidence: electron paramagnetic resonance evidence used in manual assertion
Supporting statements:
These results demonstrate that the polypeptide α helix forms distinct hydrophobic and electrostatic contacts with the DPC micelles, and are in agreement with the SDSL/EPR mapping and positioning of the α-helical model of this epitope of MBP on the surface of a lipid bilayer [6,7].
Protein: DP00236 (UniProt P02686)
Region: residues 215	to 230
Annotation: lipid binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In aqueous solution (pure water, and 100 mm KCl, pH 6.5), the spectra indicated that the polypeptide had little or no regular secondary structure. In organic and membrane-mimetic conditions (30% TFE and 20 mm DPC, respectively), the spectra clearly indicated an α-helical conformation.
Protein: DP00238 (UniProt P38398)
Region: residues 100	to 1649
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains.
NMR spectroscopy was used to characterize the structure of the central region. 1H–15N HSQC spectra were acquired for fragments #2–#21. As shown in Figure 2(A) and Supplementary Figure 1, all 20 proteins had spectra that reflected soluble, non-aggregated, but disordered proteins. Each fragment has very little chemical shift dispersion in the 1H dimension of the HSQC spectrum, and the peak widths were narrow, both features of disordered proteins.
1H–15N HSQC NMR spectra. The majority of the peaks are clustered in between 7.5 ppm and 8.5 ppm in the 1H dimension, indicating an unfolded protein.
These data taken together suggest that a large portion of the central region of BRCA1 does not contain stable, independently folded domains in the absence of binding partners.
Due to the difficulties in purifying large amounts of the full-length BRCA149 for biophysical analysis, we have designed 20 soluble fragments of BRCA1 and used three biophysical assays to demonstrate that fragments #2–#21 encompassing the central region of BRCA1 are mostly intrinsically disordered, with the possibility of having some small secondary structural elements.
Protein: DP00238 (UniProt P38398)
Region: residues 100	to 1649
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains.
In order to test for the presence of secondary structure, circular dichroism (CD) spectroscopy was next utilized. Again, most fragments had similar CD spectra reflective of very little secondary structure. Most fragments had a slight minimum near 220 nm (mean residue ellipticity (MRE)∼3000) suggestive of a very small amount of α-helix and a much stronger minimum near 200 nm reflecting a predominantly disordered protein (Figure 2(B) and Supplementary Figure 2).
Circular dichroism spectra. Weak signal at 220 nm may reflect a small amount of helical content, but the majority of these peptides are disordered as evidenced by the minimum near 203–208 nm.
These data taken together suggest that a large portion of the central region of BRCA1 does not contain stable, independently folded domains in the absence of binding partners.
Due to the difficulties in purifying large amounts of the full-length BRCA149 for biophysical analysis, we have designed 20 soluble fragments of BRCA1 and used three biophysical assays to demonstrate that fragments #2–#21 encompassing the central region of BRCA1 are mostly intrinsically disordered, with the possibility of having some small secondary structural elements.
Protein: DP00238 (UniProt P38398)
Region: residues 100	to 1649
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains.
As a final test for the existence of folded globular domains, limited proteolysis was performed to see if there were any stable, protease-resistant domains in the central region. Most constructs were degraded by trypsin within minutes compared to a positive control using malate synthase, a globular protein which took more than 21 hours to start to degrade under the same conditions (Figure 2(C) and Supplementary Figure 3).
SDS-PAGE of aliquots from a proteolysis time course monitored over 21 hours after incubation with trypsin. In contrast to the globular enzyme malate synthase which is largely resistant to protease digestion for more than 21 hours (Supplementary Figure 3(A)), these constructs are digested by trypsin within ten minutes.
These data taken together suggest that a large portion of the central region of BRCA1 does not contain stable, independently folded domains in the absence of binding partners.
Due to the difficulties in purifying large amounts of the full-length BRCA149 for biophysical analysis, we have designed 20 soluble fragments of BRCA1 and used three biophysical assays to demonstrate that fragments #2–#21 encompassing the central region of BRCA1 are mostly intrinsically disordered, with the possibility of having some small secondary structural elements.