INITIAL HYDROPHOBIC COLLAPSE IN THE FOLDING OF BARSTAR

Two models are commonly used to describe the poorly understood earliest steps of protein folding. The framework model(1-3) stresses very early formation of nascent secondary structures, which coalesce into a compact, molten, globule-like form(4,5) from which structure slowly develops(6,7). The hydrophobic collapse model(8-10) gives overriding precedence to a nonspecific collapse of the polypeptide chain which facilitates 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 dye 8-anilino-1-naphthalene-sulphonic acid on binding, and restoration of tryptophan-dansyl fluorescence energy transfer as structure-monitoring probes. We show that the polypeptide chain rapidly collapses (within 4 ms) to a compact globule with a solvent-accessible hydrophobic core(6), but with no optically active secondary or tertiary structure. Thus the earliest event of the major folding pathway of barstar is a nonspecific hydrophobic collapse that does not involve concomitant secondary structure formation.