Figure 2: Structural characterization of the hydrophobic effect in protein folding.

(a–c) Number of hydrogen bonds per water molecule for the CDS (272 K), NS (298 K) and HDS (323 K). The number of hydrogen bonds is calculated for bulk water molecules (BWM, farther than 0.4 nm), and for interface water molecules (IWM, water molecules in the first shell around the protein, within 0.4 nm) considering both the water-water hydrogen bonds (light blue) and the water-protein hydrogen bonds (blue). In our calculations, we found that bulk and interface water molecules form the same number of hydrogen bonds at the three temperatures. (d–f) At cold denaturation temperatures water molecules win the competition with the protein to form hydrogen bonds with the protein hydrogen bond donors and acceptors. Two hydrogen bonds formed by a hydrophobic residue are shown in particular, for which only the backbone amide and carbonyl groups can form hydrogen bonds; these two water-protein hydrogen bonds are highlighted in blue. By contrast, under folding conditions intra-protein hydrogen bonds are preferentially formed, and no particular preference is present at hot denaturation temperatures; the two protein-protein hydrogen bonds (within a α-helix) are highlighted in yellow. (g) Number of hydrogen bonds formed by frataxin in the CDS, NS and HDS ensembles. The number of protein-protein hydrogen bonds is shown in yellow, and that of protein-water hydrogen bonds in blue. (h) Water rotational anisotropy as a function of the distance from the protein surface for the CDS (dotted blue), NS (solid black) and HDS (dashed red), calculated as <3cos²ϑ(r) −1 > RDF(r), where ϑ is the angle of the vector from a solvent oxygen to a protein atom with the normal of the solvent plane averaged over all the solvent molecules and all the protein atoms, and RDF(r) is the non-normalised radial distribution function of the water with respect to the protein surface. (i) Total water rotational anisotropy in presence of the protein calculated as the integral of the absolute value of the former curves. The degree of order of the water molecules near the protein correspond to the extent to which they form hydrogen bonds with the protein, which is large at cold denaturing conditions, small under folding conditions, and intermediate at hot denaturing conditions.