Synthetic model proteins: contribution of hydrophobic residues and disulfide bonds to protein stability

Abstract

The objective of this study was to evaluate quantitatively the contribution that hydrophobic residues and disulfide bonds make to protein stability using a model protein with well-defined secondary, tertiary and quaternary structure. This de novo-designed protein consists of two identical 35-residue alpha-helical polypeptide chains arranged in a coiled-coil structure, which are stabilized by nine pairs of interchain hydrophobic interactions from leucine residues and an interchain disulfide bond. The mutant proteins differ from the Leu-protein only at positions 16 and 19 of each chain which contain either Ile, Val, Ala, Phe or Tyr instead of Leu residues. The stability of each protein was determined in its reduced and oxidized form from guanidine hydrochloride denaturation experiments using circular dichroism measurements. The Leu-, Ile-, Val- and Phe-proteins containing the disulfide bond were essentially 100% alpha-helical in benign medium (0.05 M phosphate buffer, pH 7, containing 0.1 M KCl) with transition midpoints of 5.3, 4.1, 2.9 and 2.4 M denaturant, respectively. Reduction of the disulfide bond had little effect on the Leu-protein but resulted in significant decreases in helicity of the other mutants. The order of protein stability of these analogs remains the same in the absence or presence of the disulfide bond (Leu- greater than Ile- greater than Val- greater than Phe- greater than Tyr- greater than Ala-protein). Interestingly, the more stable the protein was in the absence of the disulfide bond, the larger the contribution the disulfide bond made to protein stability. The identical mutation in the disulfide bridge protein had a much greater effect on protein stability than in the reduced protein. This suggests that the disulfide bond controls the conformation of the coiled-coil some 14-17 residues further along the polypeptide chain (the disulfide bond is between positions 2 and 2' and the closest mutation site in the coiled-coil is at position 16 and 16'). In contrast, in the absence of the disulfide bond, the coiled-coil is more flexible and can accommodate the mutations more easily by adjusting the interchain packing around the mutation sites. The helix-forming tendency of nonpolar residues was deduced in this study by comparing the alpha-helicity of the mutant polypeptides in aqueous buffer containing 50% trifluoroethanol where they exist as single-stranded helices. Hydrophobicity is an important factor in determining the alpha-helix forming tendency of aliphatic nonpolar residues in amphipathic alpha-helices.