Absolute hydration free energies of blocked amino acids: implications for protein solvation and stability

Abstract

Most proteins perform their function in aqueous solution. The interactions with water determine the stability of proteins and the desolvation costs of ligand binding or membrane insertion. However, because of experimental restrictions, absolute solvation free energies of proteins or amino acids are not available. Instead, solvation free energies are estimated based on side chain analog data. This approach implies that the contributions to free energy differences are additive, and it has often been employed for estimating folding or binding free energies. However, it is not clear how much the additivity assumption affects the reliability of the resulting data. Here, we use molecular dynamics-based free energy simulations to calculate absolute hydration free energies for 15 N-acetyl-methylamide amino acids with neutral side chains. By comparing our results with solvation free energies for side chain analogs, we demonstrate that estimates of solvation free energies of full amino acids based on group-additive methods are systematically too negative and completely overestimate the hydrophobicity of glycine. The largest deviation of additive protocols using side chain analog data was 6.7 kcal/mol; on average, the deviation was 4 kcal/mol. We briefly discuss a simple way to alleviate the errors incurred by using side chain analog data and point out the implications of our findings for the field of biophysics and implicit solvent models. To support our results and conclusions, we calculate relative protein stabilities for selected point mutations, yielding a root-mean-square deviation from experimental results of 0.8 kcal/mol.

Figure 1

Comparison of blocked amino-acid solvation free energies with their corresponding side chain analog data (kcal/mol). (Amino acids) Results for the blocked amino acids reported in Table 2 relative to Glycine. (Side chain analogs) Side chain analog results of Shirts et al. (46) relative to the side chain analog of Glycine (H₂). The approximate horizontal position of each amino acid is indicated by its one-letter code. The computational results relative to Gly ($\Delta A_{\text{amino acids}}-\Delta A_{\text{Gly}}$) or its side chain analog H₂ ($\Delta A_{\text{side}\text{chain analogs}}^{\text{Shirts}}-\Delta A_{{\text{H}}_2}$; y axis) are plotted against the experimental solvation free energy differences of the side chain analogs reported by Wolfenden et al. (14), also relative to H₂ ($\Delta A_{\text{side}\text{chain analogs}}^{\text{Wolfenden}}-\Delta A_{{\text{H}}_2}$; x axis). If the contributions of side chain and backbone to the solvation free energy were purely additive, both lines should be identical.