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Biochemistry. 1994 May 17;33(19):5867-76.

The refolding of human lysozyme: a comparison with the structurally homologous hen lysozyme.

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1
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford.

Abstract

Pulsed hydrogen exchange labeling has been used in conjunction with circular dichroism in the near and far UV to study the refolding of human lysozyme from its guanidinium chloride denatured state. Human lysozyme differs in sequence by 51 residues and one insertion from the hen protein, which has previously been studied under identical conditions by similar methods [Radford, S. E., Dobson, C. M., & Evans, P. A. (1992) Nature 358, 302-307]. The two proteins show marked differences in their folding kinetics. First, the overall rate of refolding of human lysozyme is 4-fold faster than that of the hen protein. Second, although protection of amides in the alpha-domain develops faster than that of amides in the beta-domain in both proteins, unlike hen lysozyme stabilization of the secondary structural elements of the alpha-domain in human lysozyme does not occur in a fully cooperative manner. Rather, amide hydrogens in two alpha-helices located near to the N-terminus and in the 3(10) helix close to the C-terminus of the protein are protected from exchange significantly faster than those in the remaining two alpha-helices in the alpha-domain of the protein. Third, stopped flow CD measurements show that both proteins develop extensive secondary structure during the dead time of these experiments (ca. 2 ms); this is accompanied by formation of tertiary interactions, probably involving tryptophan residues, only in the human enzyme. These results suggest that although the fundamental folding process is similar in the two proteins, human lysozyme differs in that it forms a stable subdomain involving the two N-terminal alpha-helices and the C-terminal 3(10) helix in the first few milliseconds of folding, and that at least some tryptophan residues are ordered before the formation of the native state. This indicates that the details of the folding of homologous proteins may differ as a consequence of amino acid substitutions and suggests that the study of mutant and variant proteins can provide clues as to the determinants of folding.

PMID:
8180215
[Indexed for MEDLINE]

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