Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of HIV-I protease tethered dimer by real time NMR spectroscopy

Abstract

HIV I protease has been the target of extensive and variety of investigations in recent years because of its importance in the AIDS viral life cycle. We describe here real time NMR studies on the unfolding kinetics of two tryptophans, W6 and W42, which are located in the dimerization and hinge domains of the protein, respectively. Unfolding seems to get initiated in the dimerization domain. The kinetic data at two temperatures, 32 and 42°C, can both be described by two-state models for both the tryptophans, and the final state reached at 42°C does not depend on the path of unfolding. Unfolding free energy changes derived from the kinetic fitting parameters are less than 3 kJ/mol, indicating that the energy landscape is very shallow. The free energy values and the rates for the two tryptophans are different at 32°C, but are nearly the same at 42°C. These are interpreted in the light of the "new view" of protein folding and the relative behaviors of the two tryptophans suggest the existence of cooperative pathways in the unfolding reaction of the protein. These observations would provide valuable insights into protein function, stability, and effects of nonactive site mutations conferring drug resistance.