| 光谱学与光谱分析 |
|
|
|
|
|
| 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.
|
|
Received: 2009-08-10
Accepted: 2009-11-16
|
|
|
|
Corresponding Authors:
FENG Juan
E-mail: fengjuan@uestc.edu.cn
|
|
[1] Herzog M, Dorne A M, Grellet F. Plant Mol. Biol., 1995, 27: 743.
[2] Roxrud I, Lid S E, Fletcher J C, et al. Plant Cell. Physiol., 2007, 48(3): 471.
[3] Taylor B H, Scheuring C F. Mol. and General Genetics, 1994, 243(2): 148.
[4] Medina-Escobar N, Cardenas J, Valpuesta V, et al. Anal. Biochem., 1997, 248: 288.
[5] de la Fuente J I, Amaya I, Castillejo C, et al. J. Experimental Bot., 2006, 57(10): 2401.
[6] Schunmann P HD , Smithm R C, Langm V, et al. Plant Cell and Environ., 1997 , 20: 1439.
[7] Berrocal-Lobo M, Segura A, Moreno M, et al. Plant Physiol., 2002, 128: 951.
[8] Segura A, Moreno M, Madueno F, et al. Mol. Plant-Microbe-Interaction, 1999, 12(1): 16.
[9] Kovalskaya N, Hammond R W. Protein Expression and Purification, 2009, 63: 12.
[10] Wigoda N, Ben-Nissan G, Granot D, et al. Plant J., 2006, 48(5): 796.
[11] Peng J Z, Lai L J, Wang X J. Sci. China, Ser. C-Life Sci., 2008, 51(6): 520.
[12] ZHOU Jian-ping, YANG Zu-jun, BAI Li-li, et al(周建平，杨足君，白莉莉，等). Acta Botanica Boreali-Occidentalia Sinica(西北植物学报), 2006, 26(9): 1874.
[13] LIU Yuan, MENG Guo-quan, ZHOU Jian-ping, et al(刘 渊，蒙国权，周建平，等). J. Agr. Biotechnology(农业生物技术学报), 2009, 17(3): 510.
[14] TANG Wei-hua, ZHANG Jing-liu, WANG Zong-yang(唐威华，张景六，王宗阳). Bot. Physiol. Acta(植物生理学报), 2000, 26(1): 64.
[15] YUAN Yan, XIAO Han, KANG Hong-jun, et al(袁 燕，肖 涵，康洪钧，等). Spectroscopy and Spectral Analysis(光谱学与光谱分析), 2009, 29(3): 777.
|
| [1] |
ZHANG Jin-wei1, 2, CAO Nian1, 2, CHEN Wu-yong1, 2*. Influence of Microwave Irradiation on Collagen Triple Helix Structure[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2018, 38(05): 1353-1357. |
| [2] |
LI Qing, WANG Xiang-hui*, DU Juan, ZHANG Jie, CHEN Shu-de, QIAO Deng-jiang . Effect of 33 Hz Pulsed Electric Field on Insulin Amyloidosis [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2016, 36(04): 1069-1074. |
| [3] |
XI Jia-fu1, TANG Lei1*, ZHANG Jian-hua2, ZHANG Hong-jian2, CHEN Xu-sheng2, MAO Zhong-gui2 . Characterization of Structural Change of Ascorbate Peroxidase from Chinese Kale during Denaturation by Circular Dichroism[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2014, 34(11): 3062-3065. |
| [4] |
WEN Peng-cheng1, 2，YU Dan-dan3，WANG Xin-xin1，ZHANG Lu-da2，REN Fa-zheng2，LEI Xin-gen4，GUO Hui-yuan2* . A Comparison of Different Treatment Conditions on the Conformation Changes of Bovine Lactoferrin [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2012, 32(01): 162-165. |
| [5] |
WU Xu-li1，2, ZHU Qian-qian3，CHENG Xiao-juan3, XU Hong3, LIU Zhi-gang2, WU Hui1* . Spectroscopic Studies on the Relationship Between Heat Treatment Process and Immunogenicity of β-Lactoglobulin in Milk [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2011, 31(08): 2205-2209. |
| [6] |
ZHANG Fa-sheng1,3, LIU Zuo-xin1*, WAN Hao-lei2,3, LIU Miao1 . Fractal Characteristics of Visible Spectra Across a Hilly Area[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2011, 31(02): 473-477. |
| [7] |
CHEN Hua1, WANG Li2. Investigation of Guanidine Hydrochloride Induced Chlorophylls Denaturation by Terahertz Time-Domain Spectroscopy[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(10): 2619-2621. |
| [8] |
LU Yan, ZHANG Wei-wei, WANG Gong-ke. Progress in Study of Secondary Structure of Denaturized Protein by FTIR[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2008, 28(01): 88-93. |
| [9] |
ZHU Jian-qi1, LIU Zhi-gang1*, HUANG Hai-zhen2, ZHU Yong-feng1, XU Hong1 . Study of Fluorescence Spectra of Blattela Germanica Allergen, Derenatured and Renatured Bla g 2[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2006, 26(12): 2254-2257. |
| [10] |
HU Jin-mei1, WEI Dong1*, GUO Si-yuan1, CHEN Feng1,2 . Effects of Environmental Factors and Denaturants on the Specific Absorption Spectrum and Fluorescence Characters of B-Phycoerythrin[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2006, 26(12): 2258-2263. |
| [11] |
DONG Rui-xin1，2, YAN Xun-ling1, JIANG Shan2, LIU Mei-jie2, LIU Sheng-gang2 . Temperature Dependence of Collagen by Raman Spectra [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2004, 24(11): 1367-1369. |
| [12] |
DONG Rui-xin1，2, YAN Xun-ling1, WU Jing2, ZHANG An-ying2 . Temperature Dependence Study of Calf Thymus DNA by Raman Spectra [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2004, 24(09): 1075-1078. |
|
|
|
|
