Effects of redox buffer properties on the folding of a disulfide-containing protein: dependence upon pH, thiol p Ka, and thiol concentration

Abstract

Aliphatic thiols are effective as redox buffers for folding non-native disulfide-containing proteins into their native state at high pH values (8.0–8.5) but not at neutral pH values (6–7.5). In developing more efficient and flexible redox buffers, a series of aromatic thiols was analyzed for its ability to fold scrambled ribonuclease A (sRNase A). At equivalent pH values, the aromatic thiols folded sRNase A 10–23 times faster at pH 6.0, 7–12 times faster at pH 7.0, and 5–8 times faster at pH 7.7 than the standard aliphatic thiol glutathione. Similar correlations between thiol p K a values and folding rates at each pH value suggest that the apparent folding rate constants ( k app) are a function of the redox buffer properties (pH, thiol p K a and [RSH]). Fitting the observed data to a three-variable model (log k app = −4.216(± 0.030) + 0.5816(± 0.0036)pH − 0.233(± 0.004)p K a + log(1 − e −0.98 (±0.02)[RSH]) ) gave good statistics: r 2 = 0.915, s = 0.10.