Báo cáo khoa học: Acceleration of disulfide-coupled protein folding using glutathione derivatives

Acceleration of disulfide-coupled protein folding using

glutathione derivatives

Masaki Okumura1,2, Masatoshi Saiki2,3, Hiroshi Yamaguchi1 and Yuji Hidaka2

1 School of Science and Technology, Kwansei Gakuin University, Hyogo, Japan

2 Graduate School of Science and Engineering, Kinki University, Osaka, Japan

3 Department of Applied Chemistry, Faculty of Engineering, Tokyo University of Science, Yamaguchi, Japan

Introduction

The formation of the correct disulfide bonds and the

conversion of a protein into its native conformation

are the result of reversible thiol(SH)⁄ disulfide(SS)

exchange reactions that occur during protein folding

and are thermodynamically and kinetically related to

the redox potential in the biological environment. Glu￾tathione (c-Glu-Cys-Gly), one of the most abundant

thiol compounds found in cells, plays a major role in

the formation of disulfide bonds in proteins in the

endoplasmic reticulum [1]. Oxidized glutathione

(GSSG) functions as an oxidant in the formation of

disulfide bonds in proteins and reduced glutathione

(GSH) functions as a reducing agent that cleaves mis￾bridged disulfide bonds in proteins, resulting in the

formation of the thermodynamically stable conforma￾tion of proteins in vivo [2]. Because of this, glutathione

Keywords

arginine; disulfide; folding; glutathione;

uroguanylin

Correspondence

Y. Hidaka, Graduate School of Science and

Engineering, Kinki University, 3-4-1

Kowakae, Higashi-Osaka, Osaka 577-8502,

Japan

Fax: +81 6 6723 2721

Tel: +81 6 6721 2332

E-mail: [email protected]

(Received 20 October 2010, revised 18

January 2011, accepted 28 January 2011)

doi:10.1111/j.1742-4658.2011.08039.x

Protein folding occurs simultaneously with disulfide bond formation. In

general, the in vitro folding proteins containing disulfide bond(s) is carried

out in the presence of redox reagents, such as glutathione, to permit native

disulfide pairing to occur. It is well known that the formation of a disulfide

bond and the correct tertiary structure of a target protein are strongly

affected by the redox reagent used. However, little is known concerning the

role of each amino acid residue of the redox reagent, such as glutathione.

Therefore, we prepared glutathione derivatives – glutamyl-cysteinyl-argi￾nine (ECR) and arginyl-cysteinyl-glycine (RCG) – and examined their abil￾ity to facilitate protein folding using lysozyme and prouroguanylin as

model proteins. When the reduced and oxidized forms of RCG were used,

folding recovery was greater than that for a typical glutathione redox sys￾tem. This was particularly true when high protein concentrations were

employed, whereas folding recovery using ECR was similar to that of the

glutathione redox system. Kinetic analyses of the oxidative folding of prou￾roguanylin revealed that the folding velocity (KRCG = 3.69 · 10)3 s

)1

)

using reduced RCG ⁄ oxidized RCG was approximately threefold higher

than that using reduced glutathione ⁄ oxidized glutathione. In addition, fold￾ing experiments using only the oxidized form of RCG or glutathione indi￾cated that prouroguanylin was converted to the native conformation more

efficiently in the case of RCG, compared with glutathione. The findings

indicate that a positively charged redox molecule is preferred to accelerate

disulfide-exchange reactions and that the RCG system is effective in medi￾ating the formation of native disulfide bonds in proteins.

Abbreviation

Arg-C, arginylendopeptidase C; ECR, glutamyl-cysteinyl-arginine; ECRox, oxidized ECR; ECRred, reduced ECR; GSH, reduced glutathione;

GSSG, oxidized glutathione; RCG, arginyl-cysteinyl-glycine; RCGox, oxidized RCG; RCGred, reduced RCG.

FEBS Journal 278 (2011) 1137–1144 ª 2011 The Authors Journal compilation ª 2011 FEBS 1137