INITIAL HYDROPHOBIC COLLAPSE IN THE FOLDING OF BARSTAR

Two models are commonly used to describe the poorly understood earlies t steps of protein folding. The framework model(1-3) stresses very ear ly formation of nascent secondary structures, which coalesce into a co mpact, molten, globule-like form(4,5) from which structure slowly deve lops(6,7). The hydrophobic collapse model(8-10) gives overriding prece dence to a nonspecific collapse of the polypeptide chain which facilit ates subsequent formation of specific secondary and tertiary structure (11,12). Here we report our analysis of the earliest observable events of the major folding pathway of barstar, a small protein. We compare the kinetics of folding using circular dichroism at 222 nm and 270 nm, intrinsic tryptophan fluorescence, fluorescence of the hydrophobic dy e 8-anilino-1-naphthalene-sulphonic acid on binding, and restoration o f tryptophan-dansyl fluorescence energy transfer as structure-monitori ng probes. We show that the polypeptide chain rapidly collapses (withi n 4 ms) to a compact globule with a solvent-accessible hydrophobic cor e(6), but with no optically active secondary or tertiary structure. Th us the earliest event of the major folding pathway of barstar is a non specific hydrophobic collapse that does not involve concomitant second ary structure formation.