LiteFold/DisProt huggingface dataset:
Protein: DP04016 (UniProt Q9SIN3)
Region: residues 1	to 635
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Simultaneous static and dynamic light scattering (SLS/DLS) measurements were conducted to determine the apparent average single particle mass (Mapp) and the apparent average hydrodynamic radius (RS) of the proteins in solution as a function of concentration (Figure S4, [30]). Absolute molecular mass and RS were obtained by extrapolation of apparent molecular mass and RS to infinite dilution. All investigated LEA proteins are essentially monomeric under fully hydrated conditions. Compared to globular proteins, RS of all investigated LEA proteins was rather large, indicating their expanded, non-compact nature.
Protein: DP04016 (UniProt Q9SIN3)
Region: residues 1	to 635
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
We therefore monitored secondary structure of the six LEA_4 proteins in response to decreasing relative humidity (RH) using the Amide I band of Fourier-transform infrared (FTIR) spectra (Figure 6). This absorbance band mainly results from the C=O stretching vibration and is directly related to the protein backbone conformation. 
A maximum at 1650–1640 cm-1 indicated that all proteins were mainly disordered at 100% RH, which is in line with the CD data.
Protein: DP04016 (UniProt Q9SIN3)
Region: residues 1	to 635
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Figure S5 shows FTIR spectra of the six LEA_4 proteins in EG concentrations ranging from 0 to 12 osM. Similar to decreasing RH, increasing osmolarity induces coil-helix transitions, indicated by a shift of the Amide I maxima to higher wavenumbers.
Protein: DP04016 (UniProt Q9SIN3)
Region: residues 1	to 635
Annotation: response to cold (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Therefore, we analyzed the stability of liposomes modeling the lipid composition of inner chloroplast membranes (ICMM) after a freeze/thaw cycle and a dehydration/rehydration cycle in the ethylene glycolpresence of all six LEA_4 proteins at different LEA protein: lipid mass ratios (Figure 13).
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
CD spectroscopic analysis was carried out to probe the secondary structure of the proteins. The purified proteins (PpDHNA and deletion mutants Y11D11, Y6D6, Y6DM6, Y1K, K, and Y1) were analyzed by CD. As shown in Figure 2, A–G, all
protein variants (the uppermost black curve in each panel) were found to be disordered in an aqueous buffer.
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: protein stabilization (Biological process)
Evidence: enzymatic activity assay evidence used in manual assertion
Supporting statements:
Upon high-temperature treatment at 54 °C for 10 min, LDH lost most of its activity, retaining only about 5% of its initial activity (100%). However, the presence of a minimal concentration of ∼1250 nM of PpDHNA (1:4 molar ratio of LDH:additive protein) could restore its original activity.
The protective activity increased as the molar ratios of the additive proteins were increased (Fig. 3B).
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: protein stabilization (Biological process)
Evidence: Congo red staining evidence
Supporting statements:
LDH loses its activity at high-temperature forming aggregates, which could be visualized by staining with Congo red (40). LDH was found to lose its maximum activity (∼95%) at 54 °C when incubated for 10 min. LDH showed substantial aggregation as compared with the control (Fig. 3D). The results obtained were also quantified fluorometrically (Fig. 3E). However, in presence of PpDHNA, the aggregate formation was significantly less.
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: protein stabilization (Biological process)
Evidence: protein separation evidence used in manual assertion
Supporting statements:
The control cells showed a higher amount of protein in the pellet fraction as compared with the supernatant. However, E. coli cells overexpressing PpDHNA protein showed a higher amount of protein in the supernatant (211 μg) (Fig. 4C).
The presence of the proteins in the soluble fraction under temperature stress conditions correlated well with the cell survivability dataset. These data highlight the importance of the D-segment in imparting high-temperature tolerance through proteome protection.
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: response to heat (Biological process)
Evidence: cell viability assay evidence used in manual assertion
Supporting statements:
However, E. coli cells overexpressing D-segment bearing proteins PpDHNA, Y11D11, and Y6D6 showed better cell viability of 77%, 57%, and 42%, respectively.
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: multicellular organismal response to stress (Biological process)
Evidence: genetic transformation evidence used in manual assertion
Supporting statements:
D-segments enhanced the stress tolerance in transformed tobacco plants
Upon stress treatment, PpDHNA, Y11D11, and Y6D6 showed lesser wilting conditions and recovered quickly after the stress was removed.
Upon stress, the chlorophyll content (Fig. 5B) of the transformed plants decreased in the order PpDHNA, Y11D11, Y6D6 and the rest (Y6DM6, Y1K, K, and Y1) showed similar values. Thus, the first three coped with stress the best. This result correlated with the soluble sugar and proline content, both indicators of how good the plant is coping with stress.
Protein: DP04017 (UniProt Q6UNL8)
Region: residues 1	to 554
Annotation: protein homodimerization activity (Molecular function)
Evidence: yellow fluorescent protein fusion protein localization evidence used in manual assertion
Supporting statements:
To investigate whether the DHN molecule can self-interact and to understand the role of amphipathic α-helices for the same, a bimolecular fluorescence complementation (BiFC) assay was performed. The analysis showed a strong YFP signal, localized at the plasma membrane transiently coexpressing PpDHNA/PpDHNA and Y11D11/Y11D11 (Fig. 7, A, B, and J).
These results indicated that PpDHNA could self-associate with amphipathic α-helices, whether its K- or D-segment, being the major contributor behind such interactions.
Protein: DP04018 (UniProt Q9LW12)
Region: residues 1	to 225
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Figure S5 shows FTIR spectra of the six LEA_4 proteins in EG concentrations ranging from 0 to 12 osM. Similar to decreasing RH, increasing osmolarity induces coil-helix transitions, indicated by a shift of the Amide I maxima to higher wavenumbers.
Protein: DP04018 (UniProt Q9LW12)
Region: residues 1	to 225
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
All proteins showed typical random coil spectra in dilute solution. With increasing concentrations of each of the co-solvents, the changes in the spectral shape report on the formation of ordered secondary structure.
Protein: DP04018 (UniProt Q9LW12)
Region: residues 1	to 225
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Simultaneous static and dynamic light scattering (SLS/DLS) measurements were conducted to determine the apparent average single particle mass (Mapp) and the apparent average hydrodynamic radius (RS) of the proteins in solution as a function of concentration (Figure S4, [30]). Absolute molecular mass and RS were obtained by extrapolation of apparent molecular mass and RS to infinite dilution. All investigated LEA proteins are essentially monomeric under fully hydrated conditions. Compared to globular proteins, RS of all investigated LEA proteins was rather large, indicating their expanded, non-compact nature.
Protein: DP04018 (UniProt Q9LW12)
Region: residues 1	to 225
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
We therefore monitored secondary structure of the six LEA_4 proteins in response to decreasing relative humidity (RH) using the Amide I band of Fourier-transform infrared (FTIR) spectra (Figure 6). This absorbance band mainly results from the C=O stretching vibration and is directly related to the protein backbone conformation. A maximum at 1650–1640 cm-1 indicated that all proteins were mainly disordered at 100% RH, which is in line with the CD data.
Protein: DP04018 (UniProt Q9LW12)
Region: residues 1	to 225
Annotation: response to cold (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Therefore, we analyzed the stability of liposomes modeling the lipid composition of inner chloroplast membranes (ICMM) after a freeze/thaw cycle and a dehydration/rehydration cycle in the ethylene glycolpresence of all six LEA_4 proteins at different LEA protein: lipid mass ratios (Figure 13).
Protein: DP04019 (UniProt Q9T0H6)
Region: residues 1	to 109
Annotation: disorder (Structural state)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
We therefore monitored secondary structure of the six LEA_4 proteins in response to decreasing relative humidity (RH) using the Amide I band of Fourier-transform infrared (FTIR) spectra (Figure 6). This absorbance band mainly results from the C=O stretching vibration and is directly related to the protein backbone conformation. A maximum at 1650–1640 cm-1 indicated that all proteins were mainly disordered at 100% RH, which is in line with the CD data.
Protein: DP04019 (UniProt Q9T0H6)
Region: residues 1	to 109
Annotation: disorder (Structural state)
Evidence: dynamic light scattering assay evidence used in manual assertion
Supporting statements:
Simultaneous static and dynamic light scattering (SLS/DLS) measurements were conducted to determine the apparent average single particle mass (Mapp) and the apparent average hydrodynamic radius (RS) of the proteins in solution as a function of concentration (Figure S4, [30]). Absolute molecular mass and RS were obtained by extrapolation of apparent molecular mass and RS to infinite dilution. All investigated LEA proteins are essentially monomeric under fully hydrated conditions. Compared to globular proteins, RS of all investigated LEA proteins was rather large, indicating their expanded, non-compact nature.
Protein: DP04019 (UniProt Q9T0H6)
Region: residues 1	to 109
Annotation: disorder to order (Structural transition)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
All proteins showed typical random coil spectra in dilute solution. With increasing concentrations of each of the co-solvents, the changes in the spectral shape report on the formation of ordered secondary structure.
Protein: DP04019 (UniProt Q9T0H6)
Region: residues 1	to 109
Annotation: disorder to order (Structural transition)
Evidence: Fourier-transform infrared spectroscopy evidence used in manual assertion
Supporting statements:
Figure S5 shows FTIR spectra of the six LEA_4 proteins in EG concentrations ranging from 0 to 12 osM. Similar to decreasing RH, increasing osmolarity induces coil-helix transitions, indicated by a shift of the Amide I maxima to higher wavenumbers.
Protein: DP04019 (UniProt Q9T0H6)
Region: residues 1	to 109
Annotation: response to cold (Biological process)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Therefore, we analyzed the stability of liposomes modeling the lipid composition of inner chloroplast membranes (ICMM) after a freeze/thaw cycle and a dehydration/rehydration cycle in the ethylene glycolpresence of all six LEA_4 proteins at different LEA protein: lipid mass ratios (Figure 13).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
The CD spectra of MsDHN1 showed a minimal peak close to 209 nm (Figure S3), and contained a considerable amount of random coil structure (accounting for 65% of the total). These are typical characteristics of intrinsically disordered proteins (IDPs), which have a large negative peak at approximately 200 nm and a high ratio of coil conformation (Receveur-Brechot et al., 2005). These results demonstrate that MsDHN1 is a highly disordered protein.
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein homodimerization activity (Molecular function)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
Circular dichroism (CD) analysis showed that a negative band occurred at 228 nm in the CD spectrum, which supported our finding that MsDHN1
formed dimers in vitro (Figure S3).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein homodimerization activity (Molecular function)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
In order to demonstrate that this MsDHN1 dimerization also occurs in plants, a
bimolecular fluorescence complementation (BiFC) analysis was performed in tobacco leaves. Fluorescence signals were detected in the cytoplasm when MsDHN1-cYFP (the C-terminal fragment of YFP) and MsDHN1-nYFP (the Nterminal fragment of YFP) co-transformed in tobacco leaves (Figure 1k), indicating that the MsDHN1 protein is able to interact with itself and form homodimers in plant cells.
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein homodimerization activity (Molecular function)
Evidence: sodium dodecyl sulfate polyacrylamide gel electrophoresis evidence used in manual assertion
Supporting statements:
The results showed that the 50-kD polypeptide was an MsDHN1 homodimer (Figure 1j).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: cytosol (Cellular component)
Evidence: fluorescence evidence used in manual assertion
Supporting statements:
Unexpectedly, MsDHN1 protein showed only a cytoplasmic localization in epidermal cells of tobacco leaves or in cells of hairy roots (Figure 1b–f).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: multicellular organismal response to stress (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Compared with the WT or the vector line, growth rates of hairy roots (Figure 2c,d) or plant seedlings (Figures 2e and S5) were significant higher in MsDHN1-OE lines and lower in MsDHN1-RNAi lines under Al stress, respectively. These results indicate that MsDHN1 positively affects alfalfa tolerance to Al stress.
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein binding (Molecular function)
Evidence: glutathione S-transferase pull-down assay evidence used in manual assertion
Supporting statements:
We applied the pull-down approach to identify putative MsDHN1-interacting proteins to elucidate the molecular basis for MsDHN1 influence on oxalic acid secretion.
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein binding (Molecular function)
Evidence: yeast 2-hybrid evidence used in manual assertion
Supporting statements:
Then, a series of yeast-two-hybrid (Y2H) assays was implemented between MsDHN1 and candidate proteins. Two positive clones (MsPIP2;1 and MsTIP1;1) that encoded AQP were found finally (Figure S9).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein binding (Molecular function)
Evidence: protein fragment functional complementation evidence used in manual assertion
Supporting statements:
BiFC assays were performed to verify the MsDHN1-MsPIP2;1 or MsTIP1;1 interaction in plant cells. When MsDHN1-cYFP was transiently co-expressed with MsPIP2;1/MsTIP1;1-nYFP in Nicotiana benthamiana leaf cells, reconstituted YFP fluorescence was observed in the cytomembrane. Similarly, YFP fluorescence was observed when MsDHN1-nYFP co-expressed with MsPIP2;1/MsTIP1;1-cYFP (Figure 4b).
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: protein binding (Molecular function)
Evidence: co-immunoprecipitation evidence used in manual assertion
Supporting statements:
Co-immunoprecipitation (Co-IP) assays further demonstrated that MsDHN1 associated with AQPs in N. benthamiana leaf cells (Figure 4c). These results suggest that MsDHN1 interacts with AQPs in plant.
Protein: DP04020 (UniProt A0A7G5VWW1)
Region: residues 1	to 222
Annotation: stress response to metal ion (Biological process)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Organic acid has the function of chelating Al to alleviate Al toxicity in plants
Compared with the WT or the vector line, significantly increased oxalate exudation was observed from root tips exposed to 100 μm AlCl3 in MsDHN1-OE lines (Figure 3g,i); in contrast, significantly decreased oxalate exudation was observed in MsDHN1-RNAi lines (Figures 3h and S7).
MsDHN1 modulates Al accumulations in root tips via oxalic acid secretion
Protein: DP04022 (UniProt Q6BDI9)
Region: residues 1	to 14
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No electron density was observed
for the first 14 amino acids, the middle 117 to 130 amino acids
and for the last residue.
Cross-references: PDB:8A4A
Protein: DP04022 (UniProt Q6BDI9)
Region: residues 117	to 130
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No electron density was observed
for the first 14 amino acids, the middle 117 to 130 amino acids
and for the last residue.
Cross-references: PDB:8A4A
Protein: DP04023 (UniProt P10949)
Region: residues 10	to 25
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No
electron density was observed for the first 16 amino acids and the
last 30 amino acids of Rab3CGppNHP_Q222H_10-227 (bovine).
Cross-references: PDB:8A4A
Protein: DP04023 (UniProt P10949)
Region: residues 198	to 227
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No
electron density was observed for the first 16 amino acids and the
last 30 amino acids of Rab3CGppNHP_Q222H_10-227 (bovine).
Cross-references: PDB:8A4A
Protein: DP04024 (UniProt P63040)
Region: residues 71	to 134
Annotation: disorder (Structural state)
Evidence: far-UV circular dichroism evidence used in manual assertion
Supporting statements:
In the absence of lipids, the CD spectrum of the
mCpx1 CTD is consistent with the protein being
largely disordered in solution, but a slight
decrease in signal around 220 nm that indicates
the unbound protein contains some helical
structure (Figure 3(A)).
Protein: DP04024 (UniProt P63040)
Region: residues 71	to 134
Annotation: disorder (Structural state)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
These observations are further corroborated by
ps-ns backbone relaxation data collected on both
free and micelle-bound mCpx1 CTD. In the
absence of micelles,15N R1 and R2
measurements, along with heteronuclear {1H-15N}
NOE (hetNOE) data are consistent with the
protein being disordered in solution (Figure 4, S4).
Cross-references: BMRB:51396
Protein: DP04024 (UniProt P63040)
Region: residues 114	to 134
Annotation: lipid binding (Molecular function)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
Comparison of 2D {15N-1H} HSQC spectra for free and DPC-bound mCpx1 CTD revealed that spectroscopic changes between the two states are limited to specific residues in the protein (Figure 2(A)).
Residues that are significantly affected by the presence of micelles all fall in the C-terminus of the CTD, specifically the terminal ∼ 20 residues (114–134). These residues exhibit both chemical shift perturbations (CSPs) (Figure 2(B)) and attenuated signal intensities (Figure 2(C)) in the presence of micelles. Together, these data suggest that the C-terminus of the mCpx1 CTD is interacting with DPC-micelles while the N-terminal regions of the CTD are largely unaffected.
Comparison of 2D {15N-1H} HSQC spectra for the mCpx1 CTD free or bound to SUVs with two different lipid compositions (60% DOPC/25% DOPE/15% DOPS and 85% POPC/15% POPS) revealed significant attenuation of peak heights in the presence of SUVs (Figure 5(A)), without detectable chemical shift changes. There is a global decrease in signal intensity for all peaks in the SUV-bound spectra, but the C-terminal 20 residues show a greater decrease in intensity for both SUV compositions (Figure 5(B)) indicating that this region is interacting strongly with SUVs irrespective of their composition.
Cross-references: BMRB:51397
Protein: DP04024 (UniProt P63040)
Region: residues 114	to 134
Annotation: disorder to order (Structural transition)
Evidence: nuclear magnetic resonance spectroscopy evidence used in manual assertion
Supporting statements:
In the presence of DPC-micelles, two distinct regions in the C-terminus of the mCpx1 CTD, encompassing residues 116–124 and 128–133, form stable helical structure (Figure 3(C), Figure S3(B-C)) that is not observed in the absence of micelles.
Residues spanning the regions from ∼ 114–124 and 128–133 showed clear NH-NH NOEs from residue i to i ± 1, characteristic of helical structure, consistent with the regions defined as helical in the TALOS-N predictions. Residues 125–127 consist of Pro-Gly-Pro, precluding the observation of NH-NH i to i + 1 NOEs. However, this sequence is highly unfavorable for helical structure and likely represents a break between the two helices, as indicated by the chemical shift-based prediction.
Notably, residues 125–127, constituting the non-helical PGP linker between the AH- and CT-motif helices, does not exhibit indications of increase mobility, suggesting that in the micelle-bound conformation, the linker conformation, while non-helical, may be relatively fixed.
Cross-references: BMRB:51397
Protein: DP04026 (UniProt P53228)
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:
The final
model displayed excellent geometry except for the first
eleven amino acids (amino acids 1–11 at the N-terminus,
both in molecule A and B) and a region of an outside
loop (203–205 in molecule A and 202–206 in molecule
B), which were disordered in the crystal.
Cross-references: PDB:3CQ0
Protein: DP04027 (UniProt P37837)
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:
In the electron density maps, the polypeptide chain is well defined except for 10 residues at the N-terminus and five residues at the C-terminus.
Cross-references: PDB:1F05