Abstract
Understanding the dominant factor in thermodynamic stability of proteins remains an open challenge. Kauzmann’s hydrophobic interaction hypothesis, which considers hydrophobic interactions between nonpolar groups as the dominant factor, has been widely accepted for about sixty years and attracted many scientists. The hypothesis, however, has not been verified or disproved because it is difficult, both theoretically and experimentally, to quantify the solvent effects on the free energy change in protein folding. Here, we developed a computational method for extracting the dominant factor behind thermodynamic stability of proteins and applied it to a small, designed protein, chignolin. The resulting free energy profile quantitatively agreed with the molecular dynamics simulations. Decomposition of the free energy profile indicated that intramolecular interactions predominantly stabilized collapsed conformations, whereas solvent-induced interactions, including hydrophobic ones, destabilized them. These results obtained for chignolin were consistent with the site-directed mutagenesis and calorimetry experiments for globular proteins with hydrophobic interior cores.
Highlights
Understanding the dominant factor behind thermodynamic stability of proteins remains a challenging issue in biochemistry, biophysics, and molecular biology[1,2,3]
In 1959, Kauzmann concluded in his seminal review[8] that hydrophobic attraction was a dominant factor in the thermodynamic stability of the folded conformation for many globular proteins. This has been supported by the following experimental observations: (i) the change in Gibbs energy for transferring a small nonpolar molecule from an aqueous solution to an organic solvent is large and negative[8]; (ii) the net effect of electrostatic interactions on protein stability is negligibly small[9]; and (iii) numerous nonpolar residues are located in the interior of globular proteins[10,11]
It was shown that (1) both hydrophobic interactions[12] and intramolecular hydrogen bonding[13] contributed substantially to protein stability; (2) the enhancement of van der Waals interactions due to tight packing in the protein interior caused by the replacement of small hydrophobic residues with larger ones resulted in increased protein stability[14]; and (3) the effect of hydrogen bonding of peptide groups on protein stability was comparable to that of hydrogen bonding of side chains[13,15]
Summary
Understanding the dominant factor behind thermodynamic stability of proteins remains a challenging issue in biochemistry, biophysics, and molecular biology[1,2,3]. In 1959, Kauzmann concluded in his seminal review[8] that hydrophobic attraction was a dominant factor in the thermodynamic stability of the folded conformation for many globular proteins This has been supported by the following experimental observations: (i) the change in Gibbs energy for transferring a small nonpolar molecule from an aqueous solution to an organic solvent is large and negative[8]; (ii) the net effect of electrostatic interactions on protein stability is negligibly small[9]; and (iii) numerous nonpolar residues are located in the interior of globular proteins[10,11]. Μnonpol(R) is expected to provide the upper limit of the contribution of the solvent-induced hydrophobic interaction to the hydrophobic collapse of the protein since μnonpol(R) includes the nonpolar contributions to μex(R) arising from all polar residues as well Such a decomposition of F(R) should provide insights into understanding the dominant factor in the thermodynamic stability of the protein. We present an efficient computational method to evaluate the free energy profile and the components as a function of a coordinate R using a combination of continuum solvent MD simulations and a recently developed reference-modified density functional theory (RMDFT) for calculation of solvation free energy[29,30,31,32]
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