Molten globule unfolding monitored by hydrogen exchange in urea.

Abstract

The molten globule state of Escherichia coli ribonuclease H1 was studied by hydrogen exchange in order to understand how the energetics of individual regions react to the presence of denaturant. Hydrogen exchange rates were monitored (1) directly by NMR spectroscopy and (2) indirectly by quenching the exchange process and returning to the native state 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 experiments were modeled after the recently developed "native state hydrogen exchange" experiment and were carried out as a function of urea concentration. The free energy of hydrogen exchange varied linearly with 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 of unfolding measured by hydrogen exchange and corresponding m values varied for each residue implying a noncooperative molten globule structure. These results are in contrast to similar exchange experiments on native proteins which generally display more than one type of exchange behavior. The single type of exchange seen in the molten globule is probably due to its larger conformational freedom and noncooperative nature.