Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.
Equilibrium and kinetic measurements of the conformational transition of reduced thioredoxin.
复制标题
还原型硫氧还蛋白构象转变的平衡和动力学测量。
DOI：
10.1021/bi00379a029
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Stellwagen,E
中科院分区：
文献类型：
--
作者：
Kelley,RF;Shalongo,W;Jagannadham,MV;Stellwagen,E
爱荷华州爱荷华州市，爱荷华州医学院生物化学系，1986年6月9日接收; 1986年11月5日接收修订的Mandarin摘要：在中性pH和25 ℃下，通过与20倍过量的还原二硫苏糖醇反应，大肠杆菌硫氧还蛋白中的单个二硫键被还原.对于一些测量，还原的硫氧还蛋白进一步与碘乙酰胺反应以烷基化半胱氨酰残基。用远紫外圆二色性和排阻色谱法研究了氧化型、还原型和还原型烷基化硫氧还蛋白的变性转变。二硫键的断裂使天然硫氧还蛋白对变性的稳定性降低约2.4 kcal/mol，随后的烷基化使稳定性进一步降低1.6 kcal/mol。本文用排阻色谱法在2 ℃、中压条件下研究了还原态硫氧还蛋白在盐酸胍中的构象变化动力学。单一和多重混合协议的分析是一致的，在变性状态和一个紧凑的nativelike中间体在复性过程中的瞬态积累的一个占主导地位的非本地配置。该中间体可以包含非天然构型并且可以适应其异构化。没有令人信服的色谱证据被发现的构象具有洗脱时间不同于天然或变性蛋白质的特征。大肠杆菌氧化硫氧还蛋白的晶体学模型（Holmgren等人，1975年）表明，这项研究得到了普通医学研究所的美国公共卫生服务研究基金GM-22109、心肺血液研究所的计划项目基金HL-14388以及国家科学基金会的赠款PCM-8313046和DMB-8413658的支持。
Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242 Received June 9, 1986; Revised Manuscript Received November 5, 1986 abstract: The single disulfide bond in Escherichia coli thioredoxin was reduced by reaction with a 20-fold excess of reduced dithiothreitol at neutral pH and 25 C. For some measurements, reduced thioredoxin was further reacted with iodoacetamide to alkylate the cysteinyl residues. The denaturation transitions of oxidized, reduced, and reduced alkylated thioredoxin were observed by using far-ultraviolet circular dichroic and exclusion chromatographic measurements. Cleavage of the disulfide bond lowers the stability of the native thioredoxin to denaturation by about 2.4 kcal/mol, and subsequent alkylation lowers the stability by a further 1.6 kcal/mol. Thekinetics of the conformational change of reduced thioredoxin in guanidine hydrochloride were observed by using exclusion chromatography at moderate pressure and 2 C. Analyses of single and multimixing protocols are consistent with a predominant nonnative configuration in the denatured state and thetransient accumulation of a compact nativelike intermediate during refolding. The intermediate can incorporate the nonnative configuration and can accommodate its isomerization. No compelling chromatographic evidence was found for a conformation having an elution time different from that characteristic for either the native or the denatured protein. e crystallographic model of Escherichia coli oxidized thioredoxin (Holmgren et al., 1975) indicates that the single tThis investigation was supported by US Public Health Service Research Grant GM-22109 from the Institute of General Medical Sci-ences, by Program Project Grant HL-14388 from the Heart, Lung, and Blood Institute, and by Grants PCM-8313046 and DMB-8413658 from the National Science Foundation.