Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D2O

FEBS Letters

Goldberg, Michel E.¹  Friguet, Bertrand¹  Blond-Elguindi, Sylvie¹ 

[1] Unité de Biochimie des Régulations Cellulaires, Département de Biochimie et Génétique Moléculaire, Institut Pasteur, 25-28, rue du Docteur Roux, F-75724 Paris Cédex 15, France

关键词: Stopped-flow kinetics;    Deuterium oxide;    Tryptophan synthase;    Protein folding;    Early intermediate;    β2;    the β2-subunit of E. coli tryptophan synthase (L-serine hydrolyase (adding indole) EC 4.2.1.20);    I-AEDANS;    N-iodoacetyl-N′-(5-sulpho-1-naphthyl)ethylenediamine;    pyridoxal-P;    pyridoxal-5′-phosphate;    GdnHCl;    guanidinium chloride;    β2-AEDANS;    β2 labelled with I-AEDANS on cysteine 170;    reduced holo-β2;    β2 carrying the sodium borohydride reduced Schiff base between pyridoxal-P and the ϵ-amino group of lysine 87;   

DOI  :  10.1016/0014-5793(88)81071-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Guanidine hydrochloride (GdnHCl) at high concentrations, e.g. 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins. The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50–100-fold in a ‘renaturation buffer’. Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing. Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows. Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D₂O) to adjust the density of the renaturation buffer. This enabled us to study the appearance of a short-lived intermediate during the folding of the β₂-subunit of Escherichia coli tryptophan synthase.

【 授权许可】

Unknown