Concentration-dependent hydrogen exchange kinetics of 3H-labeled S-peptide in ribonuclease S

Schreier, A.A.; Baldwin, R.L.

Journal of Molecular Biology 105(3): 409-426


ISSN/ISBN: 0022-2836
PMID: 9516
DOI: 10.1016/0022-2836(76)90101-7
Accession: 068517864

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The H exchange kinetics of the S-peptide in RNase S can be measured by 1st tritiating the S-peptide in the absence of S-protein and then allowing it to recombine rapidly with S-protein. Afterwards the exchange reactions of this specific segment of RNase S can be studied. The exchange kinetics of bound S-peptide are complex, indicating that different H+ exchange at markedly different rates. The terminal exchange reaction, involving at least 5 highly protected H+, was studied as a function of pH. At low concentrations of RNase S the exchange kinetics become concentration-dependent, owing to the dissociation of the S-peptide. Although the fraction of free S-peptide is always very small, its rate of exchange is several orders of magnitude faster than that of bound S-peptide, and the concentration dependence of the exchange kinetics is readily measurable. It provides a highly sensitive method for determining small KD. Values of KD ranging from 10-6 M at pH 2.7, C, to 2 .times. 10-10 M at pH 7.0, C, are reported. The value for KD at pH 7.0, C, confirms the data and extrapolation to C of Hearn et al. At high concentrations of RNase S the terminal exchange reaction is independent of concentration. It probably results from a local unfolding reaction of the bound S-peptide. Above pH 4 the strong pH dependence of KD closely resembles that of the apparent equilibrium constant for this local unfolding reaction. The latter may be 1 step in the dissociation process. Such a model is presented for RNase S dissociation. Measurement of concentration-dependent exchange kinetics should provide a useful method of determining small Kd in other systems, e.g., in studies of protein-nucleic acid interactions.