Temperature dependence of water interactions with the amide carbonyls of α-helices.

Protein folding, stability, and function depend on the properties of water in complex ways that are not completely understood. The influence of solvent arises from the interplay between the water and protein structures and their dynamics, including entropic effects, disruption of water structure, solvation of hydrophobic groups, and hydrogen bonding interactions.1,2 The hydrophobic effect is thought to be a key driving force of protein folding, because it favors the folded structure in which hydrophobic residues are sequestered from solvent within the hydrophobic core of the protein. Consequently, understanding the interactions between aqueous solvent and proteins, including hydrophobic hydration, has been the subject of numerous experimental and theoretical studies.3-16 Several general principles have emerged from this work. The free energy change associated with the hydration of hydrophobic molecules is governed primarily by the associated changes in water structure.17 The temperature dependence of the hydrophobic effect parallels the temperature-dependent solubility of hydrophobic or nonpolar molecules. The solubility of hydrophobic molecules in water initially decreases with increasing temperature, followed by an increase in solubility with increasing temperature, resulting in a minimum in solubility, usually above room temperature.1 More insight into the nature of the interaction of water with proteins has come from theoretical studies, including density functional theory (DFT) calculations3-6 and molecular dynamics (MD) simulations.7-9,11,18 Such studies provide a wealth of molecular detail about the interaction of water with proteins, but the conclusions have been difficult to validate experimentally. Infrared spectroscopy is an established method for the study of protein conformation,3,4,12-16,19-22 but it also has the potential to elucidate protein–solvent interactions. The amide I band in particular is sensitive to hydrogen bonding within the protein and between the protein and the aqueous solvent.12-16,23-25 An important example is the distinct vibrational frequencies observed for “solvated” versus “buried” helices: amide carbonyl groups involved in H-bonding within the helix and with the solvent occur at a lower frequency (~1632 cm−1) than helical amide carbonyl groups that are sequestered from solvent (~1652 cm−1) in the hydrophobic core of proteins or within a lipid bilayer.14,26,27 The direct effect of hydrogen-bonding on the amide I band is that it reduces the force constant of the individual amide oscillators relative to the non-hydrogen-bonded case. The solvent also indirectly affects the amide I band by changing conformational preferences, which in turn, results in different coupling strengths. Several theoretical studies have attempted to quantify these effects.3,4,6,28,29 An MD simulation of a solvated helical peptide found the calculated amide I band to be near 1630 cm−1 at low temperature, shifted by ~12 cm−1 toward the lower frequency compared with the buried one at room temperature.30 The simulation suggests that the major contribution to the observed frequency shift of the amide I band arises from the strong tendency for carbonyls to form additional hydrogen bonds with water even though they are internally hydrogen bonded. In the present study, we use temperature-dependent FTIR spectroscopy combined with singular value decomposition (SVD)31,32 and global fitting to probe the temperature-dependent hydration of unlabeled helical peptides and proteins in aqueous solutions. Although the IR spectral signature for solvated helical carbonyl groups is well-known, the difficulty in probing protein hydration arises in part from separating temperature-dependent protein solvation from the global thermal melt or unfolding of the peptide or protein. Our approach utilizes SVD and global fitting to separate these two processes, allowing the temperature dependence of the hydration of peptides and proteins to be investigated. Specifically, we studied the temperature dependence of a monomeric α-helix, the Fs peptide.26 This peptide serves as a model for the study of two helical proteins, the villin headpiece subdomain (HP36)12,33-37 and the villin headpiece (HP67).38,39 The data indicate a decrease in solvent interaction with the peptide backbone as the temperature is increased over the temperature range from low temperature to the start of the unfolding transition. This weakening of the backbone solvation occurs as a discrete transition, not a continuous change with increasing temperature. Furthermore, it occurs near the maximum of the temperature-dependent hydrophobic effect. Possible molecular origins of this effect are discussed with respect to previous MD simulations of the temperature-dependent solvation of the polypeptide backbone.