Interactions of non-detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation.
Non-detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21-27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N-phenyl-methyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8-anilino-1-naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB 256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.
Vuillard
L
L
Unité de Biochimie Cellulaire (CNRS URA 1129), Institut Pasteur, Paris, France.
Rabilloud
T
T
Goldberg
M E
ME
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives* / Q000378:metabolism; D002645:Chickens; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D017022:Flow Injection Analysis; D005456:Fluorescent Dyes; D007700:Kinetics; D009113:Muramidase / Q000378:metabolism*; D017510:Protein Folding; D014364:Tryptophan; D014367:Tryptophan Synthase / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D005456:Fluorescent Dyes; C114724:NDSB 256; D001622:Betaine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D009113:Muramidase; D014367:Tryptophan Synthase