Contribution of Hydrophobic Interactions to Protein Stability

Abstract

Our goal was to gain a better understanding of the contribution of hydrophobic interactions to protein stability. We measured the change in conformational stability, Δ(ΔG), for hydrophobic mutants of four proteins: villin head piece subdomain (VHP) with 36 residues, a surface protein from Borrelia burgdorferi (VlsE) with 341 residues, and two proteins previously studied in our laboratory, ribonucleases Sa and T1. We compare our results with previous studies and reach the following conclusions. 1. Hydrophobic interactions contribute less to the stability of a small protein, VHP (0.6 ± 0.3 kcal/mole per –CH2– group), than to the stability of a large protein, VlsE (1.6 ± 0.3 kcal/mol per –CH2– group). 2. Hydrophobic interactions make the major contribution to the stability of VHP (40 kcal/mol) and the major contributors are (in kcal/mol): Phe 18 (3.9), Met 13 (3.1), Phe 7 (2.9), Phe 11 (2.7), and Leu 21 (2.7). 3. Based on Δ(ΔG) values for 148 hydrophobic mutants in 13 proteins, burying a –CH2– group on folding contributes, on average, 1.1 ± 0.5 kcal/mol to protein stability. 4. The experimental Δ(ΔG) values for aliphatic side chains (Ala, Val, Ile, and Leu) are in good agreement with their ΔGtr values from water to cyclohexane. 5. For 22 proteins with 36 to 534 residues, hydrophobic interactions contribute 60 ± 4% and hydrogen bonds 40 ± 4% to protein stability. 6. Conformational entropy contributes about 2.4 kcal/mol per residue to protein instability. The globular conformation of proteins is stabilized predominately by hydrophobic interactions.

A polymetamorphic protein

“…28 Protein folding is driven in large part by hydrophobic association, and protein sequences generally require a threshold level of hydrophobicity to fold stably into a compact structure, [29][30][31][32] but simple protein models suggest that excessively hydrophobic sequences are less likely to fold uniquely. 28,[33][34][35] High or increased hydrophobicity is experimentally associated with chameleon sequences, lowered structural specificity, or outright fold changes in a number of proteins.…”

On the unyielding hydrophobic core of villin headpiece

“…Numerous studies have described the physical and thermodynamic consequences of altering the hydrophobic core of both the C-terminal subdomain [1][2][3][4][5][6]13 and more recently the N-terminal subdomain 7 of villin headpiece. In the studies of the C-terminal subdomain, it has been demonstrated that even relatively small perturbations within the hydrophobic core are of significant thermodynamic consequence.…”

“…The $3.3 kcal/mol destabilization of the L61G point mutation can be explained by the loss of the hydrophobic interface between leucine 61 and the residues comprising hydrophobic pocket ($1.1 6 0.5 kcal/mol per ACH 2 A group). 13 The loss of $3.5 kcal/mol between the L61[GL] and the wild-type construct is likely due to the conformational strain imposed by transforming the Cterminal end of helix 4 from an a-helix into p-helix. It is interesting that 3.5 kcal/mol destabilization is very close to the 3.3 kcal/mol destabilization attributable to the loss of the hydrophobic interface.…”

“…This result is in line with the work of Pace et al, which demonstrated that the L61A point mutant in HP35 (the C-terminal 35 residues of villin headpiece) was also highly destabilized relative to wildtype. 13 The L61G point mutant does not alter the hydrophobic core Despite being both thermally and thermodynamically destabilized, the 1 H-1 H NOESY spectra of HP67 L61G exhibits good peak dispersion and an abundance of cross peaks, which are signatures of well-folded proteins [Supporting Information Fig. 1(A)].…”

“…The $3.3 kcal/mol destabilization can be fully explained by the loss of the hydrophobic interface between leucine 61 and the residues comprising hydrophobic pocket ($1.1 6 0.5 kcal/mol per ACH 2 A group). 13 …”