INTERACTIONS OF NON DETERGENT SULFOBETAINES WITH EARLY FOLDING INTERMEDIATES FACILITATE IN-VITRO PROTEIN RENATURATION

Non-detergent sulfobetaines (NDSB) are a family of solubilizing and st abilizing agents for proteins. In a previous study [Goldberg, M. E., E xpert-Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Desi gn 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 mole cules facilitate protein renaturation. Stopped-flow and manual-mixing fluorescence and enzyme activity measurements:nts were used to compare the kinetics of protein folding in the presence and absence of enyl-m ethyl-N,N-dimethylammonium-propane-sulfonate (NDSB 256). Hen lysozyme and the beta 2 subunit of Escherichia coli tryptophan synthase were ch osen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryp tophan synthase occurs within less than 2.5 s after dilution in the re naturation buffer, but can be prevented by NDSB 256, only very early f olding phases (such as the formation of a loosely packed hydrophobic c ore able to bind 8-anilino-1-naphthalenesulphonic acid, and the initia l burying of tryptophan 177) are significantly altered by NDSB256; non e of the later phases is affected. Furthermore, NDSB 256 did not signi ficantly affect any of the kinetic phases observed during the refoldin g of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding pro cess anti acts by limiting the abortive interactions that could lead t o the formation of inactive aggregates.