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Equilibrium and kinetic studies on folding of canine milk lysozyme

Interdisciplinary Research Centre, K.U. Leuven Campus Kortrijk, B-8500 Kortrijk, Belgium

Reprint requests to: Herman Van Dael, Interdisciplinary Research Centre, K.U. Leuven Campus, Kortrijk, E. Sabbelaan 53, B-8500 KORTRIJK, Belgium; e-mail: Herman.Vandael{at}kulak.ac.be; fax: +32-56-246997.

Thermal and chemical unfolding studies of the calcium-binding canine lysozyme (CL) by fluorescence and circular dichroism spectroscopy show that, upon unfolding in the absence of calcium ions, a very stable equilibrium intermediate state is formed. At room temperature and pH 7.5, for example, a stable molten globule state is attained in 3 M GdnHCl. The existence of such a pure and stable intermediate state allowed us to extend classical stopped-flow fluorescence measurements that describe the transition from the native to the unfolded form, with kinetic experiments that monitor separately the transition from the unfolded to the intermediate state and from the intermediate to the native state, respectively. The overall refolding kinetics of apo-canine lysozyme are characterized by a significant drop in the fluorescence intensity during the dead time, followed by a monoexponential increase of the fluorescence with k = 3.6 s^-1. Furthermore, the results show that, unlike its drastic effect on the stability, Ca²⁺-binding only marginally affects the refolding kinetics. During the refolding process of apo-CL non-native interactions, comparable to those observed in hen egg white lysozyme, are revealed by a substantial quenching of tryptophan fluorescence. The dissection of the refolding process in two distinct steps shows that these non-native interactions only occur in the final stage of the refolding process in which the two domains match to form the native conformation.

Keywords: Protein folding; intermediate state; canine lysozyme; stopped-flow fluorescence; kinetics; molten globule

Abbreviations: CL, canine lysozyme • apo-CL, calcium-depleted form of CL • CD, circular dichroism • DSC, differential scanning calorimetry • HLY, human lysozyme • GdnHCl, guanidine hydrochloride • MeU-triNAG, 4-methylumbelliferyl-N,N',N''-triacetyl-ß-chitotriose

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