PROTEIN STABILITY PARAMETERS MEASURED BY HYDROGEN-EXCHANGE

The hydrogen exchange (HX) rates of the slowest peptide group NH hydro gens in oxidized cytochrome c (equine) are controlled by the transient global unfolding equilibrium. These rates can be measured by one-dime nsional nuclear magnetic resonance and used to determine the thermodyn amic parameters of global unfolding at mild solution conditions well b elow the melting transition. The free energy for global unfolding meas ured by hydrogen exchange can differ from values found by standard den aturation methods, most notably due to the slow cis-trans isomerizatio n of the prolyl peptide bond. This difference can be quantitatively ca lculated from basic principles. Even with these corrections, HX experi ments at low denaturant concentration measure a free energy of protein stability that rises above the usual linear extrapolation from denatu ration data, as predicted by the denaturant binding model of Tanford. (C) 1994 Wiley-Liss, Inc.