FOLDING AND DYNAMICS OF A BI-DOMAIN ENZYME

双域酶的折叠和动力学

• 批准号: 2444698
• 负责人: MARIA T MAS
• 金额: $ 28.28万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444698
• 关键词: chemical kinetics; circular dichroism; conformation; enzyme structure; fluorescence spectrometry; fluorescent dye /probe; laser spectrometry; molecular dynamics; mutant; phosphoglycerate kinase; protein engineering; protein folding; protein purification; site directed mutagenesis; stop flow technique; time resolved data

The aim of this project is to characterize the folding mechanism of a two- domain enzyme, 3-phosphoglycerate kinase (PGK). The experimental approach involves a combination of genetic engineering, steady-state and time- resolved fluorescence, and circular dichroism techniques. Time-resolved fluorescence energy transfer measurements will be carried out in order to determine the key intra- and inter-domain distances between pairs of genetically-engineered cysteines, labeled with extrinsic fluorescent probes. Genetically-engineered tryptophans will be used as intrinsic probes to monitor local and global changes during the unfolding and refolding transitions. The above approach is expected to provide a detailed characterization of the native, intermediate and unfolded states, as well as the time-sequence of the formation of (i) individual secondary structure elements, (ii) individual domains and (iii) the domain-domain interface. The long range goal of this study is to unravel information contained in the amino acid sequence that direct the folding of this enzyme.

本项目的目的是表征折叠机制的两个- 结构域酶，3-磷酸甘油酸激酶（PGK）。 实验方法 包括基因工程、稳态和时间的结合- 分辨荧光和圆二色性技术。 时间分辨 将进行荧光能量转移测量，以便 确定关键的域内和域间距离对之间的 用外源荧光标记的基因工程半胱氨酸 probes. 基因工程改造的基因组将被用作 在展开过程中监测局部和全局变化的探针， 重折叠过渡 上述方法预计将提供一个 天然、中间和未折叠状态的详细表征， 以及形成的时间顺序（i）个别中学 结构元素，（ii）单个域和（iii）域-域 接口. 这项研究的长期目标是解开信息 包含在指导这个折叠的氨基酸序列中， 酵素

项目成果

An engineered amino-terminal domain of yeast phosphoglycerate kinase with native-like structure.

具有天然样结构的酵母磷酸甘油酸激酶的工程氨基末端结构域。

• DOI: 10.1002/pro.5560060415
• 发表时间: 1997
• 期刊: Protein science : a publication of the Protein Society
• 作者: Sherman,MA;Chen,Y;Mas,MT
• 通讯作者: Mas,MT

Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.

酵母磷酸甘油酸激酶及其缺乏一种或两种天然色氨酸的突变体的平衡展开：圆二色性以及稳态和时间分辨荧光研究。

• DOI: 10.1021/bi00174a031
• 发表时间: 1994
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Szpikowska,BK;Beechem,JM;Sherman,MA;Mas,MT
• 通讯作者: Mas,MT