STOPPED-FLOW NMR MEASUREMENT OF HYDROGEN-EXCHANGE RATES IN REDUCED HORSE CYTOCHROME-C UNDER STRONGLY DESTABILIZING CONDITIONS

A procedure to measure exchange rates of fast exchanging protein amide hydrogens by time-resolved NMR spectroscopy following in situ initiat ion of the reaction by diluting a native protein solution into an exch anging deuterated buffer is described. The method has been used to mea sure exchange rates of a small set of amide hydrogens of reduced cytoc hrome c, maintained in a strictly anaerobic atmosphere, in the presenc e of an otherwise inaccessible range of guanidinium deuterochloride co ncentrations. The results for the measured protons indicate that hydro gen exchange in the unfolding transition region of cytochrome c reach the EX2 limit, but emphasize the difficulty in interpretation of the e xchange mechanism in protein hydrogen exchange studies. Comparison of free energies of structure opening for the measured hydrogens with the global unfolding free energy monitored by far-UV CD measurements has indicated the presence of at least one partially unfolded equilibrium species of reduced cytochrome c. The results provide the first report of measurement of free energy of opening of structure to exchange in t he 0-2-kcal/mol range. Proteins 32:241-247, 1908. (C) 1998 Wiley-Liss, Inc.