Effects of Redox State of Disulfide Bonds on the Intrinsic Fluorescence and Denaturation of Trx-Fused Gibberellin-Induced Cysteine-Rich Protein from Gymnadnia conopsea
ZHANG Teng1, FENG Juan1*, LI Yang1, CHEN Rui1, TANG Li-xia1, PANG Xiao-feng1, REN Zheng-long1, 2
1. School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China 2. Sichuan Province Key Laboratory of Plant Genetics and Breeding, Sichuan Agricultural University, Ya’an 625014, China
Abstract：In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni2+-NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol·L-1 GSSG or 5 mmol·L-1 peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol·L-1 DTT was absent or present, the fusion protein could not be fully unfolded with λmax<350 nm following the treatment of 6 mol·L-1 GdnHCl. (5) Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (ΔG) of 3.7 kJ·mol-1. However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.
张 腾1，冯 娟1*，李 阳1，陈 锐1，汤丽霞1，庞小峰1，任正隆1, 2 . 二硫键的氧化还原状态对Trx融合赤霉酸诱导富含半胱氨酸蛋白内源荧光及变性过程影响[J]. 光谱学与光谱分析, 2010, 30(02): 395-400.
ZHANG Teng1, FENG Juan1*, LI Yang1, CHEN Rui1, TANG Li-xia1, PANG Xiao-feng1, REN Zheng-long1, 2 . Effects of Redox State of Disulfide Bonds on the Intrinsic Fluorescence and Denaturation of Trx-Fused Gibberellin-Induced Cysteine-Rich Protein from Gymnadnia conopsea . SPECTROSCOPY AND SPECTRAL ANALYSIS, 2010, 30(02): 395-400.
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