THE PH-DEPENDENCE OF HYDROGEN-DEUTERIUM EXCHANGE IN TRP REPRESSOR - THE EXCHANGE-RATE OF AMIDE PROTONS IN PROTEINS REFLECTS TERTIARY INTERACTIONS, NOT ONLY SECONDARY STRUCTURE

The pH dependence of amide proton exchange rates have been measured fo r trp-repressor. One class of protons exchanges too fast to be measure d in these experiments. Among the protons that have measurable hydroge n-deuterium exchange rates, two additional classes may be distinguishe d. The second class of protons are in elements of secondary structure that are mostly on the surface of the protein, and exchange linearly w ith increasing base concentration (log k(ex) versus pH). The third cla ss of amide protons is characterized by much higher protection against exchange at higher pH. These protons are located in the core of the p rotein, in helices B and C. The exchange rate in the core region does not increase linearly with pH, but rather goes through a minimum aroun d pH 6. The mechanism of exchange for the slowly exchanging core proto ns is interpreted in terms of the two-process model of Hilton and Wood ward (1979, Biochemistry 18:5834-5841), i.e., exchange through both a local mechanism that does not require unfolding of the protein, and a mechanism involving global unfolding of the protein. The increase in e xchange rates at low pH is attributed to a partial unfolding of the re pressor. It is concluded that the formation of secondary structure alo ne is insufficient to account for the high protection factors seen in the core of native proteins at higher pH, and that tertiary interactio ns are essential to stabilize the structure.