Energetics and dynamics in protease inhibitor function

蛋白酶抑制剂功能的能量学和动力学

基本信息

批准号：
7924932
负责人：
DAVID P GOLDENBERG
金额：
$ 6.82万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The long term goal of this project is to elucidate the role of protein flexibility in determining the energetic and functional aspects of protein-protein interactions, particularly in the context of serine proteases and their natural protein inhibitors. The serine proteases play numerous metabolic and regulatory roles in humans and other organisms, and an important mechanism of their own regulation involves proteins that bind to their active sites with very high affinities, apparently mimicking a substrate, but without undergoing proteolysis at a significant rate. This study will focus on one member of this large class of inhibitors, bovine pancreatic trypsin inhibitor (BPTI). Although aspects of this protein have been extensively studied by biophysical and mutational methods, it is not yet known why it (or other members of the class) are so dramatically resistant to hydrolysis. Various destabilizing chemical and mutational modifications can increase the rate of cleavage, however, suggesting that the stability or rigidity of the structure when bound to the enzyme somehow prevents motions that are necessary for catalysis. The rigidity of the structure, which undergoes very little change upon binding to trypsin, is also likely to contribute to the very high stability of the enzyme-inhibitor complex. To test these hypotheses, the dynamics of several BPTI variants, both free and when bound to trypsin, will be studied by NMR relaxation methods. In addition, the thermodynamics of the interactions will be studied by calorimetric and fluorescence methods, and x-ray crystal structures will be determined for the BPTI-trypsin complexes. The combination of data from these three approaches will lead to an improved understanding of how protein flexibility, or the lack thereof, influences the regulation of a medically important class of enzymatic reactions. More generally, this work will contribute to the fundamental knowledge necessary to predict and understand the interactions among biological macromolecules, an area of rapidly increasing biomedical importance.

描述（由申请人提供）：该项目的长期目标是阐明蛋白质灵活性在确定蛋白质 - 蛋白质相互作用的能量和功能方面的作用，尤其是在丝氨酸蛋白酶及其自然蛋白抑制剂的背景下。丝氨酸蛋白酶在人类和其他生物中起许多代谢和调节作用，其自身调节的重要机制涉及蛋白质，蛋白质与其具有很高亲和力的活性位点结合，显然模仿了底物，但没有以很大的速度进行蛋白水解。这项研究将集中于这一大类抑制剂，牛胰腺胰蛋白酶抑制剂（BPTI）的一个成员。尽管该蛋白质的各个方面已经通过生物物理和突变方法进行了广泛的研究，但尚不知道为什么它（或班级的其他成员）对水解具有极大的抵抗力。但是，各种不稳定的化学和突变修饰可以提高裂解速率，这表明结构与酶结合时的稳定性或刚度在某种程度上阻止了催化所必需的运动。结构与胰蛋白酶结合后几乎没有变化的结构的刚度也可能导致酶抑制剂复合物的高稳定性。为了检验这些假设，将通过NMR弛豫方法研究几种BPTI变体的动力学，无论是自由还是与胰蛋白酶绑定在一起。另外，将通过量热和荧光方法研究相互作用的热力学，并将确定针对BPTI-Trypsin复合物的X射线晶体结构。来自这三种方法的数据的组合将导致人们对蛋白质柔韧性或缺乏蛋白质的柔韧性如何影响医学上重要的酶促反应的调节。更笼统地，这项工作将有助于预测和理解生物大分子之间相互作用的必要知识，这是生物医学重要性迅速提高的领域。

项目成果

期刊论文数量（16）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Functional and structural roles of the Cys14-Cys38 disulfide of bovine pancreatic trypsin inhibitor.

DOI：
10.1016/j.jmb.2008.07.063
发表时间：
2008-10-17
期刊：
JOURNAL OF MOLECULAR BIOLOGY
影响因子：
5.6
作者：
Zakharova, Elena;Horvath, Martin P.;Goldenberg, David P.
通讯作者：
Goldenberg, David P.

Detergent-assisted oxidative folding of delta-conotoxins.

DOI：
10.1034/j.1399-3011.2003.00048.x
发表时间：
2003-04
期刊：
The journal of peptide research : official journal of the American Peptide Society
影响因子：
0
作者：
R. D. Dela Cruz;F. Whitby;O. Buczek;G. Bulaj
通讯作者：
R. D. Dela Cruz;F. Whitby;O. Buczek;G. Bulaj

Amino acid replacement that eliminates kinetic traps in the folding pathway of pancreatic trypsin inhibitor.

氨基酸替代消除了胰蛋白酶抑制剂折叠途径中的动力学陷阱。