MOLTEN GLOBULE UNFOLDING MONITORED BY HYDROGEN-EXCHANGE IN UREA

The molten globule state of Escherichia coli ribonuclease H1 was studi ed by hydrogen exchange in order to understand how the energetics of i ndividual regions react to the presence of denaturant. Hydrogen exchan ge rates were monitored (1) directly by NMR spectroscopy and (2) indir ectly by quenching the exchange process and returning to the native sc are for NMR detection. Direct hydrogen exchange on the molten globule state demonstrated that the observed protons exchange 3-100-fold more slowly than in an unfolded peptide, The quenched hydrogen exchange exp eriments were modeled after the recently developed ''native state hydr ogen exchange'' experiment and were carried out as a function of urea concentration. The free energy of hydro en exchange varied linearly wi th denaturant concentration for all 29 measurable protons, suggesting that each amide's exchange behavior can be modeled with only one type of opening transition. The free energy elf unfolding measured by hydro gen exchange and corresponding m values varied for each residue implyi ng a noncooperative molten globule structure. These results are in con trast to similar exchange experiments on native proteins which general ly display more than one type of exchange behavior, The single type of exchange seen in the molten globule is probably due to its larger con formational freedom and noncooperative nature.