Role of individual disulfide bonds in hen lysozyme early folding steps.
To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme. Stopped-flow studies on the renaturation of these guanidine-unfolded proteins with their three disulfides intact showed that, for the three variants, the native far-UV ellipticity was regained in a burst phase within the 4-ms instrument dead-time. The transient overshoots of far-UV ellipticity and tryptophan fluorescence that follow the burst phase, as well as the kinetics of transient 8-anilino-1-naphthalene-sulfonic acid (ANS) binding, were diversely affected depending on the variant. Together with previous reports on the folding kinetics of WT lysozyme carboxymethylated on cysteines 6 and 127, detailed analysis of the kinetics showed that (1) none of the disulfide bonds were indispensable for the rapid formation (<4 ms) of the native-like secondary structure; (2) the two intra-alpha-domain disulfides (C6-C127 and C30-C115) must be simultaneously present to generate the trapped intermediate responsible for the slow folding population observed in WT lysozyme; and (3) the intra-beta-domain (C64-C80) and the inter-alphabeta-domains (C76-C94) disulfides do not affect the kinetics of formation of the trapped intermediate but are involved in its stability.
Guez
Valérie
V
Unité de Biochimie Cellulaire (Centre National de la Recherche Scientifique: CNRS URA 2185), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Roux
Pascale
P
Navon
Amiel
A
Goldberg
Michel E
ME
D000818:Animals; D002645:Chickens; D003553:Cystine; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000235:genetics; D020125:Mutation, Missense; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D003553:Cystine; D009113:Muramidase