Attempts to Delineate the Relative Contributions of Changes in Hydrophobicity and Packing to Changes in Stability of Ribonuclease S Mutants

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• 作者：Mili Das ; Bharathi Vasudeva Rao ; Sanjukta Ghosh ; and Raghavan Varadarajan
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：April 19, 2005
• 年：2005
• 卷：44
• 期：15
• 页码：5923 - 5930
• 全文大小：420K
• 年卷期：v.44,no.15(April 19, 2005)
• ISSN：1520-4995

文摘

While the hydrophobic driving force is thought to be a major contributor to protein stability,it is difficult to experimentally dissect out its contribution to the overall free energy of folding. We havemade large to small substitutions of buried hydrophobic residues at positions 8 and 13 in the peptide/protein complex, RNase-S, and have characterized the structures by X-ray crystallography. Thethermodynamics of association of these mutant S peptides with S protein was measured in the presenceof different concentrations of methanol and ethanol. The reduction in the strength of the hydrophobicdriving force in the presence of these organic solvents was estimated from surface-tension data as well asfrom the dependence of the s/gifchars/Delta.gif" BORDER=0 >C_p of protein/peptide binding on the alcohol concentration. The data indicateda decrease in the strength of the hydrophobic driving force of about 30-40% over a 0-30% range of thealcohol concentration. We observe that large to small substitutions destabilize the protein. However, theamount of destabilization, relative to the wild type, is independent of the alcohol concentration over therange of alcohol concentrations studied. The data clearly indicate that decreased stability of the mutantsis primarily due to the loss of packing interactions rather than a reduced hydrophobic driving force andsuggest a value of the hydrophobic driving force of less than 18 cal mol^-1 Ų.