Rapid and efficient production of functional Cytochrome P450 enzymatic complex fo

快速高效生产功能性细胞色素 P450 酶复合物

基本信息

批准号：
8520339
负责人：
Ayyalusamy Ramamoorthy
金额：
$ 28.81万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-30 至 2016-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8520339
关键词：
Binding Proteins Biochemical Biological Biological Models Cell membrane Cell physiology Cells Circular Dichroism Complex Cytochrome P450 Cytochromes Detergents Development Disease Drug Targeting Eligibility Determination Environment Enzymes Escherichia coli Fluorescence Resonance Energy Transfer Goals Human Genome Individual Investigation Label Length Lipid Bilayers Measurement Membrane Membrane Proteins Micelles Molecular Conformation NMR Spectroscopy Oryctolagus cuniculus Outcome Outcome Study Oxidation-Reduction Oxidoreductase Pattern Phase Physiological Processes Polymers Preparation Process Production Property Proteins Research Resolution Sampling Signal Transduction Site Solutions Spin Labels Structure Techniques Testing Transmembrane Domain Water Work X-Ray Crystallography drug development experience insight membrane model mimetics mutant novel protein complex protein folding public health relevance reconstitution research study screening solid state nuclear magnetic resonance structural biology surfactant

项目摘要

DESCRIPTION (provided by applicant): Membrane proteins are essential regulators of a number of cellular and physiological processes including signaling between cells, transport across cell membranes and energy transduction processes. High-resolution structures of membrane proteins can provide insight into their function and enable the development of compounds to alter their properties for biological and biomedical purposes. While more than 30% of the human genome and 50% of known drug targets are membrane proteins, few structures of membrane proteins are known. For most membrane proteins, this is due to the lack of membrane mimetics that contain the protein in a stable, functional, and homogeneous state. In this application, we propose to develop robust approaches to produce near-native model membranes that both stabilize membrane-bound proteins or protein-protein complexes in their functional forms and will also enable structure determination at high-resolution by NMR spectroscopy. We have chosen cytochrome proteins (CytP450, CytP450-reductase (CYPOR) and Cytb5) and their complexes (P450-CYPOR and P450- b5) as model systems for optimization. Challenges posed by these membrane proteins are manifold and therefore the biochemical and biophysical approaches developed to tackle these challenges can be relatively easily extended for structural studies of other membrane proteins. To accomplish this goal, we propose to investigate new classes of mild detergents and non-detergent surfactants as well as native membrane-like bicelles that show promise in stabilizing the native fold of membrane proteins using a rapid assignment-free screening process by NMR spectroscopy. While the focus of this proposal is on the rapid optimization of sample conditions for NMR structural studies, the outcome can be extended for investigations using a variety of other biophysical techniques including EPR, FRET and X-ray crystallography. PUBLIC HEALTH RELEVANCE: The outcome of the proposed studies on the production and optimization of membrane proteins will significantly advance our ability to obtain structural and functional insights at atomic-level resolution and will aid in the development of drugs to treat various diseases.

描述（由申请人提供）：膜蛋白是许多细胞和生理过程的重要调节剂，包括细胞间的信号传导、跨细胞膜的运输和能量转导过程。膜蛋白的高分辨率结构可以深入了解其功能，并能够开发化合物来改变其特性，用于生物学和生物医学目的。虽然超过 30% 的人类基因组和 50% 的已知药物靶标是膜蛋白，但膜蛋白的结构知之甚少。对于大多数膜蛋白来说，这是由于缺乏含有稳定、功能性和均质状态的蛋白质的膜模拟物。在此应用中，我们建议开发稳健的方法来生产近乎天然的模型膜，既可以稳定膜结合蛋白或蛋白-蛋白复合物的功能形式，又可以通过核磁共振波谱进行高分辨率结构测定。我们选择细胞色素蛋白（CytP450、CytP450-还原酶（CYPOR）和 Cytb5）及其复合物（P450-CYPOR 和 P450-b5）作为优化的模型系统。这些膜蛋白带来的挑战是多方面的，因此为解决这些挑战而开发的生化和生物物理方法可以相对容易地扩展到其他膜蛋白的结构研究。为了实现这一目标，我们建议研究新型温和去污剂和非去污剂表面活性剂以及天然膜状双分子，它们在使用核磁共振波谱快速无分配筛选过程稳定膜蛋白的天然折叠方面表现出希望。虽然该提案的重点是快速优化 NMR 结构研究的样品条件，但结果可以扩展到使用各种其他生物物理技术（包括 EPR、FRET 和 X 射线晶体学）的研究。公共健康相关性：拟议的膜蛋白生产和优化研究的结果将显着提高我们在原子水平分辨率上获得结构和功能见解的能力，并将有助于开发治疗各种疾病的药物。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

A small molecule that displays marked reactivity toward copper- versus zinc-amyloid-β implicated in Alzheimer's disease.

一种小分子，对铜- 与锌-淀粉样蛋白-β 表现出明显的反应性，与阿尔茨海默病有关。

DOI：
10.1039/c3cc48473d
发表时间：
2014-05-25
期刊：
Chemical communications
影响因子：
4.9
作者：
Savelieff MG;Liu Y;Senthamarai RR;Korshavn KJ;Lee HJ;Ramamoorthy A;Lim MH
通讯作者：
Lim MH

Two-step mechanism of membrane disruption by Aýý through membrane fragmentation and pore formation.

A××通过膜破碎和孔形成来破坏膜的两步机制。

DOI：
发表时间：
2012-08-22
期刊：
影响因子：
3.4
作者：
Sciacca, Michele F M;Kotler, Samuel A;Brender, Jeffrey R;Chen, Jennifer;Lee, Dong;Ramamoorthy, Ayyalusamy
通讯作者：
Ramamoorthy, Ayyalusamy

Phosphatidylethanolamine enhances amyloid fiber-dependent membrane fragmentation.

磷脂酰乙醇胺增强淀粉样蛋白纤维依赖性膜破碎。