Aromatic Thiol pKa Effects on the Folding Rate of a Disulfide Containing Protein

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• 作者：Jonathan D. Gough ; Joseph M. Gargano ; Anthony E. Donofrio ; and Watson J. Lees
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：October 14, 2003
• 年：2003
• 卷：42
• 期：40
• 页码：11787 - 11797
• 全文大小：135K
• 年卷期：v.42,no.40(October 14, 2003)
• ISSN：1520-4995

文摘

The production of proteins via recombinant DNA technology often requires the in vitro foldingof inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitrofolding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase thefolding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relativelyrandom distribution of disulfide bonds. The importance of the thiol pK_a value was investigated by theanalysis of five para-substituted aromatic thiols with pK_a values ranging from 5.2 to 6.6. Folding wasmeasured at pH 6.0 where the pK_a value of the thiols would be higher, lower, or equal to the solution pH.Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromaticthiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, thestandard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK_a valuedecreased. Optimal conditions occurred when the concentration of protonated thiol in solution wasapproximately 2 mM, although the total thiol concentration varied considerably. The importance of theconcentration of protonated thiol in solution can be understood based on equilibrium effects. Kineticstudies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key ratedetermining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols provedto be kinetically faster and more versatile than classical aliphatic thiol redox buffers.