TERTIARY INTERACTIONS IN THE FOLDING PATHWAY OF HEN LYSOZYME - KINETIC-STUDIES USING FLUORESCENT-PROBES

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight in to the nature of intermediates detected in our recent hydrogen-exchang e labeling studies [Radford, S. E., et al. (1992) Nature 358, 302-307] , which were performed under the same conditions. Protection from exch ange results primarily from the development of stabilizing sidechain i nteractions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrins ic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hy drophobic structure, and these studies have been complemented by exper iments involving binding to a fluorescent hydrophobic dye 1-anilino-na phthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichroism , while development of a competent active site has been probed by bind ing to a competitive inhibitor bearing a fluorescent label, -methylumb elliferyl-N,N'-diacetyl-beta-chitobiose. The combination of these tech niques has enabled us to monitor the development both of the hydrophob ic core of the protein and of interactions between the two folding dom ains, If the behavior of the tryptophans is representative of the hydr ophobic residues of the protein in general, it seems that collapse is already substantial in species formed within the first few millisecond s of refolding and is highly developed in later intermediates which no netheless appear to lack many fixed tertiary interactions. Some of the details of the native structure, including the active site which is f ormed at the interface between the folding domains, develop only in th e slowest stages of folding, even though in a subset of molecules stab le native-like structure exists in both domains at an early stage of f olding. These late events probably involve fine adjustments of side-ch ain packing and formation of specific ionic interactions that occur in the native state.