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：
8309974
负责人：
Ayyalusamy Ramamoorthy
金额：
$ 29.7万
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 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8309974
关键词：
Binding Proteins Biochemical Biological Biological Models Cell membrane Cell physiology Cells Circular Dichroism Complex Cytochrome P450 Cytochromes Detergents Development Disease Drug Delivery Systems 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 Screening procedure 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 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基本调节剂，包括细胞之间的信号传导，跨细胞膜的运输以及能量转导过程。膜蛋白的高分辨率结构可以提供有关其功能的洞察力，并使化合物的开发能够改变其特性，以实现生物学和生物医学目的。尽管超过30％的人基因组和50％的已知药物靶标是膜蛋白，但膜蛋白的结构很少。对于大多数膜蛋白，这是由于缺乏膜含有蛋白质稳定，功能性和均匀状态的膜模拟物所致。在此应用中，我们建议开发出可靠的方法，以产生近本机模型膜，以稳定膜结合的蛋白质或蛋白质 - 蛋白质络合物的功能形式，并将通过NMR光谱法以高分辨率确定结构。我们选择了细胞色素蛋白（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。这些膜蛋白提出的挑战是多种多样的，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，我们建议使用NMR Spectroscopicy通过快速的无分配筛查过程来研究新的轻度洗涤剂和非局部表面活性剂以及类似膜的双层双层。尽管该建议的重点是用于NMR结构研究样本条件的快速优化，但可以使用各种其他生物物理技术（包括EPR，FRET和X射线晶体学）进行研究，以进行研究。公共卫生相关性：关于膜蛋白生产和优化的拟议研究的结果将显着提高我们在原子级分辨率下获得结构和功能见解的能力，并将有助于开发用于治疗各种疾病的药物。