Equilibrium and kinetic folding of pigeon lysozyme.
In the present study, the search for a possible intermediate state in pigeon lysozyme is addressed by equilibrium and kinetic experiments using static and stopped-flow fluorescence and circular dichroism spectroscopies. In equilibrium conditions at pH 7.5, pigeon lysozyme shows no populated intermediate state in temperature- and GdnHCl-induced unfolding experiments. In the unfolding process at low pH, however, a distinct intermediate state with molten globule characteristics is observed. Ca2+ binding to the protein is found to stabilize the native state. The early folding intermediate observed in kinetic experiments corresponds to the equilibrium intermediate in that an important amount of secondary structure has already been established. Full accomplishment of native tertiary contacts is achieved in a fast exponential process with a rate constant (0.23-135 s-1) that is strongly dependent on refolding conditions. Binding experiments with the fluorescent inhibitor MeU-diNAG support these conclusions. The folding rate is not influenced by Ca2+ binding. Analysis of the refolding and unfolding kinetics determined as a function of denaturant concentration leads to a Gibbs energy profile with a rate-determining transition state between the N- and I-states. Comparison with previous results on the folding of hen egg white lysozyme emphasizes the crucial role of Trp 62 in stabilizing non-native interactions. The replacement of this residue by Tyr in pigeon lysozyme contributes to the formation of native tertiary contacts.
Haezebrouck
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Interdisciplinary Research Center, Katholieke Universiteit Leuven, Campus Kortrijk, Belgium.
Noyelle
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K
Van Dael
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D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002942:Circular Dichroism; D010856:Columbidae; D004791:Enzyme Inhibitors / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D009113:Muramidase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D001051:Apoenzymes; D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D006923:Hymecromone; C033855:4-methylumbelliferyl-N,N-diacetylchitobioside; D009113:Muramidase; D019791:Guanidine; D002118:Calcium