LiteFold/DisProt huggingface dataset:
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: protein binding (Molecular function)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
It has been shown previously that AF1 interacts with transcription factor TFIIF in vitro. Using Fourier transform infrared (FTIR), we tested whether this interaction can induce structure in the AR AF1. 
When the sum of the second-derivative spectra of AF1 and RAP74-CTD are compared with the spectrum of the AF1/RAP74-CTD mixture (Figure 2B), it is evident that the helical content is higher in the AF1/RAP74-CTD mixture than in the summation of the AF1 and RAP74-CTD spectra, suggesting that these changes are not just additive. Comparison of the second-derivative spectra of AF1 with and without RAP74-CTD present indicated a significant increase in helical content in AF1 when mixed with RAP74-CTD (Figure 3A), suggesting that interaction with RAP74-CTD leads to imposition of helical structure on the AF1 domain.
Our results indicate that the AF1 domain has little helical structure until it encounters RAP74. This interaction leads to formation of more helical conformation in AR AF1.  This appears to come as a result of the loss of β-sheet along with an accumulation of small losses of turns, bends, and coil.
Our findings herein, showing that AR AF1 adopts a more helical conformation when bound to RAP74, clearly indicate that AF1 can acquire helical structure through an induced-fit mechanism, when it encounters a specific binding partner.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: protein binding (Molecular function)
Evidence: radioisotope assay evidence used in manual assertion
Supporting statements:
To test whether induced folding of AR AF1 similarly enhanced coactivator binding, we carried out a series of “pulldown” experiments with the GST−AF1 fusion protein and the radiolabeled SRC-1 C-terminal domain (amino acids 977−1441, SRC-CTD).
Strikingly, a similar enhancement (40-fold) in the level of SRC-CTD binding was observed after preincubation of the GST−AF1 protein with RAP74-CTD.
Our data clearly show that the presence of TMAO and binding of RAP74 each induce a conformation in the AR AF1 domain, which facilitates AF1's interaction with SRC-1.
However, it should be emphasized that the main conclusion from the protein−protein interaction data is that preincubation with the binding partner TFIIF (RAP74), but not a noninteracting protein, significantly enhanced the interaction of SRC-CTD. We conclude that conditional folding of the AR AF1 domain is a prerequisite for tight binding of SRC-1 to AF1.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
As shown in Figure 4, in 3 M TMAO there is a large change in the peak at ∼1656 cm-1, consistent with greatly increased helical content.
TMAO is known to cooperatively fold intrinsically unstructured proteins (9, 31−33) , and cooperative folding of proteins is associated with “native-like” conformations ( 34, 35). This result suggests that the induced AF1 conformation(s) may be physiologically relevant.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: disorder (Structural state)
Evidence: in vitro cleavage assay evidence used in manual assertion
Supporting statements:
Native AR-AF1 was rapidly digested by protease treatment, with full-length protein completely digested by 10 to 15 min with trypsin and by 2 min with chymotrypsin, resulting in a stable fragment representing GST (Figure 1 and data not shown).
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: molten globule (Structural state)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Purified AR-AF1 was titrated with increasing amounts of ANS which resulted in a hyperbolic curve, and ANS-binding sites appeared saturated above 40 µM (Figure 4A). Incubation of AR-AF1 with ANS resulted in a significant increase in fluorescence intensity and a blue shift for the maximum emission to 465 nm (Figure 4B).
Taken together, these data indicate that AR-AF1 in a native state binds ANS and exhibits ANS binding characteristics similar to those of a well-characterized molten globule state protein.
Second, the fluorescence properties of complexes between AR-AF1 and the hydrophobic probe ANS suggest a partially folded intermediate resembling a pre-molten globule or molten globule state.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: molten globule (Structural state)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
Coomassie-stained gel of the peak fractions are shown: average elution volume (Ve) for AR-AF1 was calculated to be 57.7 ± 0.6 mL with a gel phase distribution coefficient (Kav) of 0.71 (Figure 6A and data not shown). By comparing these values with those of the standard proteins, we calculated the molecular mass and Stokes radius of AR-AF1 to be 64.7 ± 6.4 kDa and 36.4 ± 1.8 Å, respectively (Figure 6B). These values differ greatly from predicted values (Figure 6B) and strongly suggest AR-AF1 is less compact than a native protein similar in size. Uversky and colleagues have recently collated molecular mass and the Stokes radius for a range of proteins and generated “folding-state” curves for globular, unfolded, molten globule, and pre-molten globule proteins (48, 49, 53).
Figure 6C shows that the predicted molecular mass and Stokes radius for AR-AF1 are positioned on the folding curve for a globular structured protein, but the experimental calculated properties for AR-AF1 place the receptor transactivation domain with proteins in a molten globule state.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 448
Annotation: pre-molten globule (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In agreement with previous reports based on the use of other biophysical methods, (6) the 1H,15N-HSQC spectrum of AF-1* had the features expected in an ID region such as low HN chemical shift dispersion (Figure 2a).
The results that we obtained indicated the presence of two regions of high helical propensity (defined as ΔδCα–ΔδCβ > 1 ppm and SSP ≈ 0.5, corresponding to a helical propensity of 50%) which correspond to residues 185–200, in Tau-1, and 390–410, in Tau-5. Other regions of intermediate helical propensity (defined as ΔδCα–ΔδCβ ≈ 0.5 ppm and SSP ≈ 0.2) could also be identified, such as the region 230–240, in Tau-1, and 355–365 in Tau-5 (Figure 2b, c). In addition to identifying regions of helical secondary structure, the analysis of the chemical shifts also suggests that residues 144–154 and 270–290 of AF-1* adopt an extended conformation (ΔδCα–ΔδCβ ≈ −0.5 ppm and SSP ≈ −0.3) (Figure 2b, c).
The results that we obtained, presented in Figure 2d, indicate that the regions with nascent secondary structure revealed by the analysis of the 13C chemical shifts (Figure 2b) also display relatively high R2 values. These are especially high, reaching values of ca. 15 s–1, for three regions of sequence found in Tau-5 predicted to have low disorder propensity (26) and presenting, in two cases, high helical propensity.
Protein: DP00492 (UniProt P10275)
Region: residues 330	to 448
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
Our results show that the influence of even small amounts of D2O is substantial, changing the cloud point of the ID protein by more than 10°C under solution conditions typical for solution NMR experiments, and that this is likely due to a strengthening of hydrophobic interactions responsible for LCST LLPS.
We carried out 1H,15N-BEST-TROSY experiments at concentrations ranging from 25 to 600 μM and observed that the resonances corresponding to a substantial number of residues experienced small but measurable 1HN and 15N chemical shift perturbations as well as decreases in intensity (Figure 2(a)), equivalent to those observed for other phase separating ID proteins under conditions where no phase separation occurs.
The sign of the effect that is, that LCST LLPS is enhanced by heavy water is therefore not a particularly surprising result but the amplitude of the effect, instead, is: we note that the majority of above mentioned studies examined the consequences of replacing H2O with D2O, obtaining measurable but small changes, whereas here we show changes in cloud point as large as 25°C upon replacement of 50% H2O with D2O, that represents a very profound alteration of the phase diagram of an ID protein.
Protein: DP00492 (UniProt P10275)
Region: residues 330	to 448
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: differential interference contrast microscopy evidence used in manual assertion
Supporting statements:
We show how replacing even small fractions of H2O with D2O increases the propensity of this fragment to undergo liquid–liquid phase separation, likely reflecting a stabilization of the hydrophobic interactions that drive condensation.
We observed that Tau-5* indeed undergoes LLPS, as shown in Figure 1(b), forming droplets capable of fusing (Figure S1). We found however that at equivalent concentrations—in mass per volume units—the AD, composed of 558 residues, has a higher propensity to undergo LLPS than Tau-5*, that contains only 119 residues.
Our results indicate that even modest molar fractions of D2O in a buffer solution can have a substantial effect on the phase diagram of a protein undergoing LCST LLPS.
Protein: DP00492 (UniProt P10275)
Region: residues 142	to 485
Annotation: molten globule (Structural state)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
Structural flexibility and intrinsic disorder of AR-AF1 were studied using predictive algorithms and fluorescence spectroscopy under different experimental conditions and the results revealed this domain retains characteristics indicative of molten-globule or pre-molten-globule-like structures.
Using fluorescence spectroscopy, the λmax for tryptophan emission was found to be intermediate between that expected for a random coil (fully solvent-exposed) and globular structured protein (buried) and the tertiary structure of AR-AF1 could be both stabilized or destabilized by addition of TMAO and urea respectively (Figure 2). Together, these observations suggest that AR-AF1 exists in an unfolded state that is not random coil, but may resemble a molten-globule-like conformation.
Using spectroscopic techniques, we have shown that the structure of AF1 domain can be induced by incubation with TMAO or interestingly RAP74-CTD.
Protein: DP00492 (UniProt P10275)
Region: residues 144	to 450
Annotation: disorder (Structural state)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
Both structured and unstructured regions of AR contribute to the effective phase separation of AR and disordered N-terminal domain plays a predominant role.
The thermal shift results showed that AF-1 is a typical intrinsically disordered protein, which exhibited no obvious emission intensity changes during the heating process; however, incubation with ET516 induced a dose-dependent shift of AF-1 melting temperature (Extended Data Fig. 7e) behaving like a globular protein, suggesting that ET516 binds to a redistributed and stabilized conformational ensemble of AF-1.
Here we comprehensively studied the puncta-forming behavior of AR truncated proteins both in vitro and in vivo (Fig. 2), and found that all AR domains including NTD, DBD and LBD are involved in AR phase separation while the intrinsically disordered NTD plays a predominant role.
Protein: DP00492 (UniProt P10275)
Region: residues 144	to 450
Annotation: disorder to order (Structural transition)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
The thermal shift results showed that AF-1 is a typical intrinsically disordered protein, which exhibited no obvious emission intensity changes during the heating process; however, incubation with ET516 induced a dose-dependent shift of AF-1 melting temperature (Extended Data Fig. 7e) behaving like a globular protein, suggesting that ET516 binds to a redistributed and stabilized conformational ensemble of AF-1.
On the basis of the results from thermal shift experiments (Extended Data Fig. 7e) and MD simulation (Extended Data Fig. 7f), ET516 may bind to and stabilize a redistributed AF-1 conformation.
Protein: DP00492 (UniProt P10275)
Region: residues 144	to 450
Annotation: molecular condensate scaffold activity (Molecular function)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
With the aid of PEG3350 that closely simulates the crowding environment, NTD but not DBD or LBD protein, readily formed phase-separated droplets in vitro (Extended Data Fig. 3c), consistent with its predominant LLPS potential in optoDroplets system. FRAP experiments confirmed the liquid-like feature of NTD droplets in vitro (Extended Data Fig. 3d).
Both NLS-fused OptoDroplets system and in vitro LLPS experiments demonstrated that NTD, but not DBD or LBD, possesses the intrinsic LLPS potential (Fig. 2g and Extended Data Fig. 3c).
Protein: DP00492 (UniProt P10275)
Region: residues 144	to 450
Annotation: non-membrane-bounded organelle assembly (Biological process)
Evidence: microscopy evidence used in manual assertion
Supporting statements:
The optoDroplets system fused with NLS and Cry2 is effective to compare the ability of forming nuclear foci for different regions in a more tunable and consistent manner. Time-lapse images showed that blue light induced robust formation of spherical droplets of NTD, while DBD and LBD barely formed droplets (Fig. 2g).
Both NLS-fused OptoDroplets system and in vitro LLPS experiments demonstrated that NTD, but not DBD or LBD, possesses the intrinsic LLPS potential (Fig. 2g and Extended Data Fig. 3c).
Protein: DP00492 (UniProt P10275)
Region: residues 1	to 537
Annotation: disorder (Structural state)
Evidence: intrinsic fluorescence-based protein conformation evidence
Supporting statements:
The λmax for tryptophan in buffer was 342±2 nm (n=4), which is markedly red shifted to 350 nm in the presence of urea. This is consistent with the tryptophan residues becoming more solvent exposed as the polypeptide chain is unfolded; in addition, a peak was also observed at around 305 nm due to tyrosine residues, indicating less efficient quenching by energy transfer in the unfolded protein (Fig. 2C). These data are consistent with the AR-NTDQ20 having a limited amount of stable folded structure that is lost upon treatment with urea.
Protein: DP00492 (UniProt P10275)
Region: residues 1	to 537
Annotation: disorder (Structural state)
Evidence: urea-induced protein unfolding evidence used in manual assertion
Supporting statements:
The λmax for tryptophan in buffer was 342±2 nm (n=4), which is markedly red shifted to 350 nm in the presence of urea. This is consistent with the tryptophan residues becoming more solvent exposed as the polypeptide chain is unfolded; in addition, a peak was also observed at around 305 nm due to tyrosine residues, indicating less efficient quenching by energy transfer in the unfolded protein (Fig. 2C). These data are consistent with the AR-NTDQ20 having a limited amount of stable folded structure that is lost upon treatment with urea.
Figure 3B shows the loss of ANS fluorescence for each AR-NTD polypeptide with increasing concentrations of urea. All three polypeptides showed non-cooperative unfolding and AR-NTDQ45 was consistently more sensitive to urea denaturation than either the wild-type (Q20) or the AR-NTDΔQ polypeptides. The marked enhancement of ANS fluorescence on binding to the AR-NTD polypeptides taken together with the lack of cooperative unfolding by increasing concentrations of urea indicates that each polypeptide has at least some ‘molten globule’ character with limited stable tertiary structure.
Protein: DP00492 (UniProt P10275)
Region: residues 1	to 537
Annotation: disorder (Structural state)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Figure 3B shows the loss of ANS fluorescence for each AR-NTD polypeptide with increasing concentrations of urea. All three polypeptides showed non-cooperative unfolding and AR-NTDQ45 was consistently more sensitive to urea denaturation than either the wild-type (Q20) or the AR-NTDΔQ polypeptides. The marked enhancement of ANS fluorescence on binding to the AR-NTD polypeptides taken together with the lack of cooperative unfolding by increasing concentrations of urea indicates that each polypeptide has at least some ‘molten globule’ character with limited stable tertiary structure.
Protein: DP00492 (UniProt P10275)
Region: residues 1	to 537
Annotation: disorder (Structural state)
Evidence: in vitro cleavage assay evidence used in manual assertion
Supporting statements:
To investigate further the structural implications of poly-Q repeat length on the conformation of the AR-NTD, sensitivity to limited proteolysis was used as a probe for global protein structure. All three polypeptides were sensitive to digestion by endo Glu-C protease, with the full-length protein completely digested between 5 and 15 min (Fig. 4).
Trypsin treatment similarly resulted in digestion of the full-length NTD polypeptides and major N-terminal fragments of 38, 49 and 33 kDa for ARN-Q20, -Q45 and ΔQ respectively (Fig. 5B).
Protein: DP00499 (UniProt Q9Z2F5)
Region: residues 306	to 430
Annotation: disorder (Structural state)
Evidence: proton-based nuclear magnetic resonance evidence used in manual assertion
Supporting statements:
In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains.
The C-terminal region of CtBP3 displays sequence and structural features typical of intrinsically unstructured proteins, a property that may be functional to the assembly of CtBP in the core nuclear complex and/or for the recognition of diverse molecular partners.
These signals appear to be poorly resolved, mainly due to the reduced spread of amide resonances, falling all in the 7.6–8.7 ppm chemical shift range. This observation usually indicates the absence of structural organization of the backbone (Wüthrich 1986).
Protein: DP00499 (UniProt Q9Z2F5)
Region: residues 306	to 430
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains.
The C-terminal region of CtBP3 displays sequence and structural features typical of intrinsically unstructured proteins, a property that may be functional to the assembly of CtBP in the core nuclear complex and/or for the recognition of diverse molecular partners.
The difference spectrum shows a slightly positive trend in the 208–240 nm region, indicating no significant increase of α or β secondary structure in the full-length protein relative to t-CtBP3, and a clear minimum around 200 nm, in the region associated to random coil/unfolded conformation (Hollósi et al. 1993).
Protein: DP00499 (UniProt Q9Z2F5)
Region: residues 306	to 430
Annotation: disorder (Structural state)
Evidence: small-angle X-ray scattering evidence used in manual assertion
Supporting statements:
In the present communication we apply a combined approach based on bioinformatics, nuclear magnetic resonance, circular dichroism spectroscopy, and small-angle X-ray scattering, and we show that the CtBP C-terminal region is intrinsically unstructured in the full-length CtBP and in constructs lacking the substrate- and/or the nucleotide-binding domains.
The C-terminal region of CtBP3 displays sequence and structural features typical of intrinsically unstructured proteins, a property that may be functional to the assembly of CtBP in the core nuclear complex and/or for the recognition of diverse molecular partners.
The excluded volume (126 ± 10 × 103 Å3) is larger than the value expected for a dimeric construct, which may point to unusual flexibility and high degree of hydration of the overall molecular structure. Remarkably, the values of Rg (56.3 ± 0.7 Å) and Dmax (200 ± 10 Å) exceed those of tetrameric full-length CtBP3, indicating an extremely anisotropic shape for the C-term(2) construct. One can thus conclude that the latter construct must be at least partly unfolded.
Cross-references: SASBDB:SASDAE4
Protein: DP00505 (UniProt P04234)
Region: residues 127	to 171
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD analysis of CD3εcyt, CD3δcyt, CD3γcyt, FcεRIγcyt, Igαcyt, and Igβcyt showed that these proteins are unstructured, random-coiled proteins. In each case, no detectable secondary or tertiary structure was observed with increasing protein concentration (data not shown).
Protein: DP00505 (UniProt P04234)
Region: residues 127	to 171
Annotation: self-assembly (Disorder function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The gel filtration data indicated that there exists a fast dynamic equilibrium between monomeric and dimeric cytoplasmic domains of all of the ITAM-containing proteins studied, again with the exception of Igαcyt (see below).
Protein: DP00505 (UniProt P04234)
Region: residues 130	to 171
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The electron microscopy evidence of the structure of human T cell receptor-CD3 complex shows that this region lacks electron density, indicating that it is disordered.
Cross-references: PDB:6JXR
Protein: DP00505 (UniProt P04234)
Region: residues 1	to 21
Annotation: disorder (Structural state)
Evidence: cryogenic electron microscopy-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The electron microscopy evidence of the structure of human T cell receptor-CD3 complex shows that this region lacks electron density, indicating that it is disordered.
Cross-references: PDB:6JXR
Protein: DP00506 (UniProt P07766)
Region: residues 1	to 11
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Although present in the molecule crystallized, the first 11 residues of CD3-ε had no corresponding electron density.
Cross-references: PDB:1XIW
Protein: DP00506 (UniProt P07766)
Region: residues 153	to 207
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD analysis of CD3εcyt, CD3δcyt, CD3γcyt, FcεRIγcyt, Igαcyt, and Igβcyt showed that these proteins are unstructured, random-coiled proteins. In each case, no detectable secondary or tertiary structure was observed with increasing protein concentration (data not shown).
Protein: DP00506 (UniProt P07766)
Region: residues 153	to 207
Annotation: self-assembly (Disorder function)
Evidence: gel-filtration evidence used in manual assertion
Supporting statements:
The gel filtration data indicated that there exists a fast dynamic equilibrium between monomeric and dimeric cytoplasmic domains of all of the ITAM-containing proteins studied, again with the exception of Igαcyt (see below).