A schematic representation shows protein undergoes spontaneous aggregation in aqueous medium through specific conformational transformation prone to aggregation. ATP can prevent protein aggregation and improves its solubility. ATP’s effect in protein conformational plasticity has been tested for two contrasting protein molecules belonging from two extreme spectrums of the protein family. One is the globular, structurally ordered protein Trp-cage and the other one is intrinsically disordered protein (IDP), Aβ40, containing comparatively more charged residues (according to the nature of typical IDPs). The highly aggregation prone Aβ40 protein is popularly well known for causing neurodegenerative disorders (Alzheimer’s disease, AD). The structures of both the proteins are shown in the new cartoon representation highlighting the protein region wise coloration scheme. For Trp-cage the three distinct regions, 1. Helix (H1, 1-9), 2. 3-10 Helix (H2, 10-15) and 3. Coil (coil, 16-20) are shown in green, pink and navy blue colors respectively. For Aβ40, the 1. N-terminal region (NTR, 1-16), 2. central hydrophobic core (CHC, 17-21), 3. turn (TR, 24-27), 4. secondary hydrophobic region (SHR, 30-35), and the 5. C-terminal regions (CTR, 36-40) are shown in gold, purple, blue, green and red colors respectively. The hydrophobicity index of each of the proteins is shown in pie chart representation containing acidic (gold), basic (green), hydrophobic (red) and neutral (blue) residue content. Each of the proteins shown in the surface model are colored according to the respective hydrophobicity nature. Protein-ATP (base part: red, sugar moiety: cyan and phosphate group: green) site specific interactions are tested. The current study of ATP’s effect on protein conformational plasticity is performed combining both simulation and experiment based on computational predictions validated by experimental measurement followed by computational reasoning and correlation of ATP driven conformational modification to protein aggregation scenario.

A. The probability distribution of Rg of Trp-cage compared in neat water and in 0.1 and 0.5 M ATP. B. Probability of the total number of contact formation among the residues of Trp-cage monomer are compared in absence (water) and presence of ATP (at 0.1 and 0.5 M). The probability distribution of native contacts (nc) of the protein in water and in 0.1 and 0.5 M ATP solutions are being shown in figure C. Figure D, E and F represent the 2D free energy profile of Trp-cage corresponding to the Rg and nc of the protein in water, 0.1 and 0.5 M ATP solutions respectively. Snapshots containing overlay of protein’s conformations in absence of ATP (neat water) and in presence of ATP (0.5 M) are shown in figure G and H respectively. Protein is colored by secondary structure and ATP molecules in figure H are shown in line representation with an atom based coloring scheme (C: cyan, N: blue, O: red).

Residue wise total percentage of helix and 3-10 helix content of Trp-cage protein in absence and presence of ATP (0.5 M ATP) are shown in figure A and B respectively. Figure C. The solvent accessible surface area is calculated (with gromacs module of “gmx sasa”) for Trp-cage and represented for the aqueous medium without and with ATP corresponding to each of the protein residues in a bar plot representation. Figure D shows a representative snapshot of ATP’s (in licorice representation with atom based coloring scheme) interaction with Trp-cage (new cartoon representation. Green: H1 (1-9), pink: H2 (10-15), navy blue: coil (16-20)). E. Preferential interaction coefficient (Γ) of different parts of ATP: PG, sugar and base (with respect to solvent, water) with protein are being compared. F. Bar plot representation of coulombic and LJ interaction performed by all three different parts of ATP (PG, sugar and base) with Trp-cage. Figure G. represents the comparative plots of the preferential interaction coefficient (Γ) of ATP with the three structurally different parts (H1, H2 and coil) of Trp-cage. H. The change in the secondary structure content (helix and 3-10 helix) due to action of ATP are being represented. The difference in helix and 3-10 helix content in neat water from that of ATP solution are shown in bar plots.

The 2D free energy profile of Aβ40 monomer estimated with respect to Rg and total number of intra-chain contacts are shown in figure A and B for Aβ40 in neat water and 0.5 M ATP respectively. Figure C. compares the simulation snapshots of Aβ40 monomer in neat water and in presence of ATP. Multiple conformations are overlaid for each of the cases to represent the statistical significance. Protein is colored region wise as done in Figure 1 and ATP molecules are shown by gray color line representation. Figure D. compares the β-sheet content of the Aβ40 protein in water and in 0.5 M ATP solution. Figure E and F show the residue wise intra chain contact map of Aβ40 in absence and in presence of ATP (0.5 M) respectively.

Figure A, B and C show the representative snapshots of different pairs of interacting residues namely, D23-K28, V24-N27 and L17-I32 respectively compared for salt water. The similar set of interactions are being represented in Figures D, E and F for ATP solution containing salt. G. Preferential interaction coefficient (Γ) of different parts of ATP (PG, sugar and base) with protein are being represented with respect to solvent water. H. The combined coulombic and LJ interaction energies imparted by all the three parts of ATP with Aβ40 are shown. I. The free energy of solvation (calculated by the gromacs module of “gmx sasa”) of Aβ40 protein in absence and in presence of ATP are shown in a bar plot diagram. The vertical lines over the bars show the error bars.

Figure A shows the representative snapshot of the peptide belonging to the nucleating core (16th to 22nd residue [Ac-KLVFFAE-NH2, Ac-KE]) of the Aβ40 protein, which is utilized for the experimental measurements. Figure B represents emission spectra of ThT in the presence (blue) and absence (pink) of peptide assembly in 10 mM HEPES buffer pH 7.2. Excitation wavelength (λex) = 440 nm. (Final concentration [Ac-KE]=200 µM, [ThT]=30 µM). Figure C shows a comparative plot of ThT (30 µM) assay of AcKE (300 µM) assembly with time in 10 mM pH 7.2 HEPES buffer with 0 mM (blue curve), 6 mM (yellow curve) and 20 mM (dark red curve) of ATP. The vertical lines over the bars show the error bars.

Library of TEM micrographs of Ac-KE (300 µM) assemblies in 10 mM pH 7.2 HEPES buffer at 5 min (up) and after 18 h (down) of incubation, in presence of A. 0 mM ATP B. 6 mM ATP and C. 20 mM ATP are being represented.

Figure A and B show the time profile of distance between the two actively interacting regions of two protein chains namely CHC-CHC and CHC-SHR respectively both in neat water and in presence of ATP co-solute (0.5 M ATP in 50 mM NaCl solution). Figure C and D represent residue-wise inter-protein contact map of Aβ40 in water and in 0.5 M ATP solution respectively. The contacts (CHC-CHC, CHC-SHR, SHR-SHR) found in neat water are highlighted. Figure E. The preferential interaction coefficient (Γ) of ATP with each different part of Aβ40 protein (NTR, CHC, TR, SHR and CTR) are being shown. F. Interaction of Aβ40 protein chain with ATP cosolute. ATP molecules are being shown in vdw representation. G. The interacting ATP molecules crowd around the two Aβ40 protein chains are being shown. Figure H and I show the consequence of Aβ40 dimer in neat water and in presence of ATP respectively. The corresponding simulation snapshots are being shown for simulation starting with Aβ40 dimer in water and in 0.5 M ATP in 50 mM NaCl solution. Figure J represents the time profile of distance between the two protein copies of the preformed Aβ40 dimer in exposure to ATP solution (0.5 M ATP in 50 mM NaCl solution).

Figure A shows the time profile of distance between the two protein chains (CHC-CHC) in neat water (blue curve), in 0.5 M NaXS (green curve) and ATP (red curve) in 50 mM NaCl solution. Figure B represents the probability distribution of the distance between the protein chains in each of the three (above mentioned) cases. Figure C shows the percentage of bound of the proteins (for all the three systems) in a bar plot representation. Figure D depicts the total number of intermolecular contacts of the protein monomers in each of the three solutions. Figure E and F represent the difference of residue-wise inter-protein contact map of Aβ40 in 0.5 M ATP and 0.5 M NaXS solution respectively from that of the neat water system. Figure G and H show the representative snapshots captured during the Aβ40 dimerization simulation in 0.5 M NaXS and 0.5 M ATP solution respectively. Figure I represents the interaction energy between the NaXS (green bar) and ATP (red bar) molecules with the protein molecules in a bar plot representation. The vertical lines (black colored) show the error bars in the estimation.