Tertiary Interactions in the Folding Pathway of Hen Lysozyme: Kinetic Studies Using Fluorescent Probes

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Abstract

The refolding kinetics of hen lysozyme have been studied using a range of fluorescent probes. These experiments have provided new insight into the nature of intermediates detected in our recent hydrogen-exchange labeling studies [Radford, S.E., et al. (1992) Nature 358, 302-307], which were performed under the same conditions. Protection from exchange results primarily from the development of stabilizing side-chain interactions, and the fluorescence studies reported here have provided a new perspective on this aspect of the refolding process. The intrinsic fluorescence of the six tryptophan residues and its susceptibility to quenching by iodide have been used to monitor the development of hydrophobic structure, and these studies have been complemented by experiments involving binding to a fluorescent hydrophobic dye 1-anilino-naphthalenesulfonic acid (ANS). Formation of fixed tertiary interactions of aromatic residues has been monitored by near-UV circular dichorism, while development of a competent active site has been probed by binding to a competitive inhibitor bearing a fluorescent label, 4-methylumbelliferyl-N,N'-diacetyl-beta-chitobiose. The combination of these techniques has enabled us to monitor the development both of the hydrophobic core of the protein and of interactions between the two folding domains. If the behavior of the tryptophans is representative of the hydrophobic residues of the protein in general, it seems that collapse is already substantial in species formed within the first few milliseconds of refolding and is highly developed in later intermediates which nonetheless appear to lack many fixed tertiary interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

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