The molten globule intermediate of apomyoglobin and the process of protein folding

Abstract

The molten globule model for the beginning of the folding process, which originated with Kuwajima's studies of α-lactalbumin (Kuwajima, K., 1989, Proteins Struct. Funct. Genet. 6, 87-103, and references therein), states that, for those proteins that exhibit equilibrium molten globule intermediates, the molten globule is a major kinetic intermediate near the start of the folding pathway. Pulsed hydrogen-deuterium exchange measurements confirm this model for apomyoglobin (Jennings, P.A. and Wright, P.E., in prep.). The energetics of the acid-induced unfolding transition, which have been determined by fitting a minimal three-state model (N mutually implies I mutually implies U; N = native, I = molten globule intermediate, U = unfolded) show that I is more stable than U at neutral pH (Barrick, D. and Baldwin, R.L., 1993, Biochemistry 32, in press), which provides an explanation for why I is formed from U at the start of folding. Hydrogen exchange rates measured by two-dimensional NMR for individual peptide NH protons, taken together with the CD spectrum of I, indicate that moderately stable helices are present in I at the locations of the A, G, and H helices of native myoglobin (Hughson, F.M., Wright, P.E., and Baldwin, R.L., 1990, Science 249, 1544-1548). Directed mutagenesis experiments indicate that the interactions between the A, G, and H helices in I are loose (Hughson, F.M., Barrick, D., and Baldwin, R.L., 1991, Biochemistry 30, 4113-4118), which can explain why I is formed rapidly from U at the start of folding. These experiments are consistent with the explanation proposed earlier (Baldwin, R.L., 1989, Trends Biochem. Sci. 14, 291-294) for the stabilization of native secondary structures in molten globule intermediates, namely that each unit of native secondary structure has a hydrophobic face, and the hydrophobic surfaces of two units of secondary structure can interact loosely to provide mutual stabilization. These topics are discussed here in the light of some additional results.