LiteFold/DisProt huggingface dataset:
Protein: DP00251 (UniProt O88339)
Region: residues 144	to 575
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: DP00251 (UniProt O88339)
Region: residues 144	to 575
Annotation: disorder (Structural state)
Evidence: heat capacity-based 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: DP00251 (UniProt O88339)
Region: residues 144	to 575
Annotation: clathrin coat assembly (Biological process)
Evidence: electron microscopy evidence used in manual assertion
Supporting statements:
Assuming that, in epsin 1, this activity is also located in the carboxyl-terminal part of the protein, we tested fragment His6-epsin 1-(144–575). As expected, this epsin 1 fragment also possessed assembly activity, although it was somewhat lower compared with that of the full-length protein.
Protein: DP00251 (UniProt O88339)
Region: residues 144	to 575
Annotation: clathrin coat assembly (Biological process)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
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: DP00253 (UniProt P26645)
Region: residues 1	to 309
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectrum of myr MARCKS and that of non-myr MARCKS taken under physiological conditions showed single negative peaks at around 200 nm, together with small negative peaks at 220–230 nm, suggesting that the recombinant proteins have a high content of random coil structure with a small amount of α-helix.
Although myr and non-myr MARCKS showed CD spectra with similar characteristics, the intensity of the negative peak at around 220–230 nm is smaller in myr MARCKS (Fig. 1b). This suggests that the presence of a myristoyl moiety may affect part of the three-dimensional structure of MARCKS.
Protein: DP00253 (UniProt P26645)
Region: residues 1	to 309
Annotation: calmodulin binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
The addition of 150 nM recombinant myr MARCKS to 50 nM dansyl-calmodulin induced a shift in the fluorescence emission maximum of dansyl calmodulin from 510 to 490 nm as well as a 2-fold increase in the intensity at 490 nm (Fig. 2 a), suggesting that the recombinant protein retained the ability to bind to calmodulin. The dissociation constant determined by the direct fit of titration data to mass equation was 4.5±0.4 nM (n=3), a value comparable to those reported for native and recombinant protein preparations [7, 12].
Protein: DP00253 (UniProt P26645)
Region: residues 1	to 309
Annotation: calmodulin binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectra were measured with myr MARCKS mixed with calmodulin at a 1:1 ratio in the presence of Ca2+ or with the individual component alone. The CD spectra obtained with MARCKS alone or with calmodulin alone were mathematically added and the obtained spectrum was compared with the CD spectrum of the mixture (Fig. 2b). Although the depth of the large negative peak at 202 nm showed a difference, the broad peak around 220–230 nm, an indicator of the α-helical content, did not differ significantly. This implies that the full-length myr MARCKS binds to calmodulin in a non-helical structure. This is rather unusual for a calmodulin-binding protein; an increase in the α-helical content is observed upon the binding of a target protein to calmodulin. Similar results were obtained with non-myr MARCKS (data not shown).
Protein: DP00253 (UniProt P26645)
Region: residues 1	to 309
Annotation: myristoylation display site (Disorder function)
Evidence: electrospray ionization fourier transform ion cyclotron resonance mass spectrometry evidence used in manual assertion
Supporting statements:
Electrospray mass spectroscopic analysis of the recombinant non-myr MARCKS and myr MARCKS proteins indicated that the mass difference of the two proteins was 210 Da, which corresponded very well to protein myristoylation.
The recombinant myr MARCKS migrated in a 10% polyacrylamide gel with an apparent molecular mass of 80 kDa, whereas the recombinant non-myr MARCKS showed an apparent molecular mass of 68 kDa. The migrations correspond very well to those of the native myr and non-myr MARCKS proteins purified from bovine brain [16, 17]. Since the difference in mass caused by protein myristoylation is only 210 Da, the shift in migration of about 12 kDa in SDS gel may suggest a conformational change caused by the myristoyl moiety.
Protein: DP00256 (UniProt P40316)
Region: residues 1	to 110
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The lack of dispersion seen for the amide chemical shifts in the WATERGATE spectra indicates a very similar environment for all amide hydrogen atoms in the proteins and therefore a random coil state for the polypeptide. Further indication of the unfolded state of both N-terminal fragments comes from examination of the methyl proton region of the spectra. As shown in Fig. 4B, the methyl protons display little dispersion. Furthermore, the methyl proton peaks are clustered in the region of ∼1.0–0.8 ppm, whereas peaks from methyl protons in a hydrophobic environment typically are shifted upfield to ∼0.5–0.0 ppm. Thus, 1H-NMR studies strongly indicate a largely unfolded conformation for both N-terminal fragments.
Of the observable NOEs, almost all have negative values, indicating fast tumbling and local flexibility. Thus, 1H,15N NOE analysis provides little indication of stable structure.
Protein: DP00256 (UniProt P40316)
Region: residues 1	to 110
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: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The C-terminal sub-region Myc92–167 is partly helical with negative minima at 208 nm and 222 nm, while the N-terminal sub-region Myc1–88 shows little negative ellipticity around 222 nm and a minimum shifted towards 200 nm, which indicates a mainly random structure with low helical content.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 167
Annotation: molten globule (Structural state)
Evidence: dynamic fluorescence quenching evidence used in manual assertion
Supporting statements:
Myc1–167, Myc92–167 and Myc1–88 had emission maxima of 348, 342 and 351 nm, respectively, in non-denaturing buffer (Figure 5a) suggesting that the tryptophan residues are not completely buried in any of the proteins.34 All three proteins had an emission maximum at 353–355 nm under denaturing conditions, in agreement with data for unfolded proteins.34 This indicates a decrease in solvent exposure for Myc1–167 and Myc92–167 in native buffer that is in agreement with a partly folded structure, whereas the small change for Myc1–88 suggests that Myc1–88 remains unfolded, in agreement with the CD results.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 143
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In aqueous solution and over a range of temperatures, the c-myc1-143 polypeptide showed little or no secondary structure (Figure 5A). The spectra showed the characteristics of a random conformation with a minimum at around 200 nm and progressively nearing zero at wavelengths above 210 nm.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 143
Annotation: protein binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
However, the observed spectrum for the mixture is clearly distinct from the calculated additive spectra (Figure 6B). The most striking difference is the shift to the right and reduction in the minimum at around 200 nm derived from the c-myc1-143 polypeptide. Furthermore, the change in the 200 to 222 nm ratio to near unity is consistent with conversionof a random coil to an α-helical conformation.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Both 15N-R1 and {1H}-15N-nuclear Overhauser effect (NOE) relaxation of pSer62-c-Myc1–88 correspond well to results obtained for c-Myc1–88 at similar concentrations (Figures S1E–S1G), suggesting that the intrinsic disorder with transiently structured regions identified for c-Myc1–88 (Andresen et al., 2012) is maintained upon Ser62 phosphorylation.
Cross-references: BMRB:26663
BMRB:26662
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
These observations are in agreement with the formation of dynamically disordered (Forman-Kay and Mittag, 2013), or “fuzzy” (Fuxreiter and Tompa, 2012), complexes.
Binding of intact Pin1 to non-phosphorylated c-Myc1–88 results in major loss of signal intensity primarily in the conserved, transiently ordered MB0 region, but also affects the MBI region (Figures 3A and 3D). Intact Pin1 binding to pSer62-c-Myc1–88 more clearly affects the phosphorylation site and flanking residues, and perturbations extend into the transiently helical and conserved MBI region around c-Myc1–88-Trp50, while the C-terminal part remains comparatively unperturbed (Figure 3D).
Furthermore, the NMR results suggest that the conserved, transiently structured MB0 region in c-Myc1–88 is central for Pin1 interactions with c-Myc1–88 irrespective of the phosphorylation state of Ser62.
MB0 region corresponds to residues 10–35 and MBI corresponds to the 44-63 region.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: heteronuclear single quantum coherence spectroscopy evidence used in manual assertion
Supporting statements:
After extensive buffer optimization, the resolution in the HSQC spectrum of Myc-1–88 (Figure 2) is characteristic of an intrinsically disordered protein, with all amide proton shifts confined to 7.7–8.6 ppm. However, while intrinsically disordered proteins typically have uniformly sharp resonances, the Myc-1–88 spectra demonstrated heterogeneity of peak intensities and peak shapes likely reflecting an interconverting and transiently interacting heterogeneous ensemble of states.
Relaxation parameters for interpretable residues show that in the unbound state, the entire Myc-1–88 behaves as an intrinsically disordered protein with intermediate mobility (Figure 7; empty symbols). The magnitude of the heteronuclear NOEs are consistent with a disordered region, but are higher than expected for single-domain intrinsically disordered proteins. Instead, their magnitude resembles those of longer intrinsically disordered regions linked to well-folded globular domains (61,62).
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
SPR measurements showed significant binding of Pin1 to unphosphorylated c-Myc outside of the Pin1-targeted MBI (Figures 2, S2A, and S2B), and, in agreement NMR mapping of HNCO intensity ratios, suggests that Pin1 primarily binds to the MB0 region (c-Myc10–35) (Figure 3).
MBI corresponds to the 44-63 region.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 88
Annotation: disorder (Structural state)
Evidence: dynamic fluorescence quenching evidence used in manual assertion
Supporting statements:
This indicates a decrease in solvent exposure for Myc1–167 and Myc92–167 in native buffer that is in agreement with a partly folded structure, whereas the small change for Myc1–88 suggests that Myc1–88 remains unfolded, in agreement with the CD results.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 167
Annotation: molten globule (Structural state)
Evidence: circular dichroism evidence used in manual assertion
Supporting statements:
Fitting the denaturation curves to a linear extrapolation model assuming two-state unfolding33 shows that both Myc1–167 and Myc92–167 have low cooperative unfolding but that Myc1–167 has a significantly higher melting transition temperature (∼41 °C) than does Myc92–167 (∼32 °C) (Figure 4b).
The low cooperativity in unfolding is also consistent with a molten globule-like state.
Protein: DP00260 (UniProt P01106-1)
Region: residues 89	to 167
Annotation: protein binding (Molecular function)
Evidence: surface plasmon resonance evidence used in manual assertion
Supporting statements:
After immobilization, the activity of the three c-Myc surfaces was assayed by MM-1 and TBP, which have previously been shown to bind to c-Myc.18, 26 Both of the proteins showed binding to Myc92–167 and Myc1–167 (Figure 7) but no significant binding was seen to Myc1–88, not even at higher concentration of MM-1 or TBP.
Protein: DP00260 (UniProt P01106-1)
Region: residues 89	to 167
Annotation: protein binding (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Myc1–167 showed a shift in spectral intensity upon binding to MM-1 consistent with an increase in secondary structure upon binding to MM-1. A much smaller shift was observed for MM-1 with Myc92–167, whereas the spectrum of Myc1–88 with MM-1 was identical with the theoretical additive spectrum.
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 143
Annotation: protein binding (Molecular function)
Evidence: protein binding evidence used in manual assertion
Supporting statements:
A selective interaction was observed between c-myc1-143 transactivation domain and TBP and the RAP74 subunit of TFIIF (Figure 2A).
Protein: DP00260 (UniProt P01106-1)
Region: residues 1	to 143
Annotation: transcription regulator activator activity (Molecular function)
Evidence: beta-galactosidase reporter gene assay evidence used in manual assertion
Supporting statements:
Expression of wild type c-myc1-143 fused to the DBD of the glucocorticoid receptor in yeast cells resulted in strong (almost 200-fold) induction of a lac Z reporter gene driven by a single glucocorticoid response element (Figure 3A).
Protein: DP00260 (UniProt P01106-1)
Region: residues 402	to 412
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The lack of long and medium-range NOEs shows this region is disordered.
Protein: DP00260 (UniProt P01106-1)
Region: residues 363	to 381
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
The lack of long and medium-range NOEs shows this region is disordered.
Protein: DP00260 (UniProt P01106-1)
Region: residues 353	to 439
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Alone, c-Myc353–437 displayed a CD signal typical of disordered protein regions (Tompa, 2002).
IDR boundaries are 353-439 since region 437-439 is not long enough to be ordered.
Protein: DP00260 (UniProt P01106-1)
Region: residues 367	to 439
Annotation: SCF ubiquitin ligase complex binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
The shortest C-terminal fragment of c-Myc (mutant Δ2–366) was still able to bind to Skp2, suggesting that aa residues 367–439, comprising the HLH-Zip domain of c-Myc, are sufficient for Skp2 binding.
Taken together, these results suggest that the interaction between c-Myc and Skp2 requires an intact MB2 and a region in the HLH-Zip domain of c-Myc.
Protein: DP00260 (UniProt P01106-1)
Region: residues 129	to 142
Annotation: SCF ubiquitin ligase complex binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
In vitro binding studies showed that deletion of MB2 alone or in combination with MB1 reduced binding of IVT c-Myc to GST-Skp2, whereas deletion of MB1 alone had no effect (Figure 2C, left panel). Deletion of the C-terminal aa 215–439 (mutant ΔC) reduced but did not eliminate binding, whereas the combined ΔC+ΔMB2 deletion abolished the GST-Skp2 interaction.
Taken together, these results suggest that the interaction between c-Myc and Skp2 requires an intact MB2 and a region in the HLH-Zip domain of c-Myc.
MB2 comprised the region 129-142.
Protein: DP00260 (UniProt P01106-1)
Region: residues 51	to 69
Annotation: SCF ubiquitin ligase complex binding (Molecular function)
Evidence: immunoprecipitation evidence used in manual assertion
Supporting statements:
The binding of these peptides to Fbw7 was strongly dependent upon T58 phosphorylation (Fig. 3D).
These data indicate that T58 phosphorylation regulates the physical interaction of Fbw7 with the c-Myc CPD and is required for Fbw7-driven c-Myc turnover.
C-Myc CPD corresponds to the Cdc4 phosphodegron motif 55-62.
Protein: DP00260 (UniProt P01106-1)
Region: residues 55	to 68
Annotation: protein binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
We determined the structure of the complex between Bin1C−12A and a synthetic peptide, c-Myc(55-68), which includes the entire proline-rich region (Figure 5(a) and (b); and Table 1).
Cross-references: PDB:1MV0
ELM:DEG_SCF_FBW7_1
ELM:LIG_SH3_2
Protein: DP00260 (UniProt P01106-1)
Region: residues 55	to 68
Annotation: protein binding (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Both peptides were also able to interact with the Bin1CΔPxxP mutant, yielding Kd values of 18.4 μM for 12A and 9.2 μM for c-Myc.
Cross-references: PDB:1MV0
ELM:DEG_SCF_FBW7_1
ELM:LIG_SH3_2
Protein: DP00260 (UniProt P01106-1)
Region: residues 56	to 60
Annotation: phosphorylation display site (Disorder function)
Evidence: immunodetection assay evidence used in manual assertion
Supporting statements:
The His6-c-Myc substrate was shown to be phosphorylated by immunoblot analysis with antibodies that specifically recognize c-Myc phosphorylated on Thr-58 and Ser-62 (Figure 3C); the mutant His6-c-Myc(T58A/S62A) was not phosphorylated.
Protein: DP00260 (UniProt P01106-1)
Region: residues 60	to 64
Annotation: phosphorylation display site (Disorder function)
Evidence: immunodetection assay evidence used in manual assertion
Supporting statements:
The His6-c-Myc substrate was shown to be phosphorylated by immunoblot analysis with antibodies that specifically recognize c-Myc phosphorylated on Thr-58 and Ser-62 (Figure 3C); the mutant His6-c-Myc(T58A/S62A) was not phosphorylated.
Protein: DP00260 (UniProt P01106-1)
Region: residues 51	to 66
Annotation: SCF ubiquitin ligase complex binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
Recombinant Fbw7 interacted only with the phosphorylated form of the peptide, whereas Skp2, Fbw1a, Fbw2, or Fbw4 did not interact with either form (Figure 3A).
Protein: DP00262 (UniProt Q16665)
Region: residues 530	to 698
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectra for HIF-1α 530–698 and HIF-1a 403–603 (Figures 2A and 2C) lacked the typical signatures of secondary structure (negative bands at 222 and 208 nm for α helices and a negative band at 217 nm for β sheets), exhibiting instead only a small signal at 200 nm.
Protein: DP00262 (UniProt Q16665)
Region: residues 530	to 698
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
HIF-1α 530–698 and HIF-1α 403–603 eluted from the gel filtration column as well-defined peaks corresponding to Mr 95,500 and 134,700, respectively (Figure 1A), values five and six times larger than the calculated theoretical values of 18.8 and 22.3 kDa.
These results imply that both HIF-1α 530–698 and HIF-1α 403–603 have the dimensions of denatured proteins and have extended conformations.
Protein: DP00262 (UniProt Q16665)
Region: residues 530	to 698
Annotation: disorder (Structural state)
Evidence: analytical ultracentrifugation evidence used in manual assertion
Supporting statements:
Analysis of the sedimentation equilibrium centrifugation experiment, however, showed that both proteins were monomeric with a Mr of 18,000 ± 120 and 22,000 ± 500 (Figure 1B; as an example for HIF-1α 530–698).
These results imply that both HIF-1α 530–698 and HIF-1α 403–603 have the dimensions of denatured proteins and have extended conformations.
Protein: DP00262 (UniProt Q16665)
Region: residues 403	to 603
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The far-UV CD spectra for HIF-1α 530–698 and HIF-1a 403–603 (Figures 2A and 2C) lacked the typical signatures of secondary structure (negative bands at 222 and 208 nm for α helices and a negative band at 217 nm for β sheets), exhibiting instead only a small signal at 200 nm.
Protein: DP00262 (UniProt Q16665)
Region: residues 403	to 603
Annotation: disorder (Structural state)
Evidence: chromatography evidence used in manual assertion
Supporting statements:
HIF-1α 530–698 and HIF-1α 403–603 eluted from the gel filtration column as well-defined peaks corresponding to Mr 95,500 and 134,700, respectively (Figure 1A), values five and six times larger than the calculated theoretical values of 18.8 and 22.3 kDa.
These results imply that both HIF-1α 530–698 and HIF-1α 403–603 have the dimensions of denatured proteins and have extended conformations.