FEBS Letters | |
Renaturation of guanidine‐unfolded tryptophan synthase by multi‐mixing stopped‐flow dilution in D2O | |
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
【 预 览 】
Files | Size | Format | View |
---|---|---|---|
RO201912020291388ZK.pdf | 388KB | download |