Rapid and efficient production of functional Cytochrome P450 enzymatic complex fo

描述（由申请人提供）：膜蛋白是许多细胞和生理过程（包括细胞间信号传导、跨细胞膜转运和能量转导过程）的基本调节剂。膜蛋白的高分辨率结构可以提供对其功能的深入了解，并使化合物的开发能够改变其特性以用于生物和生物医学目的。虽然超过30%的人类基因组和50%的已知药物靶点是膜蛋白，但膜蛋白的结构很少。对于大多数膜蛋白，这是由于缺乏包含处于稳定、功能性和均质状态的蛋白的膜模拟物。在这个应用中，我们建议开发强大的方法来生产近天然模型膜，既稳定膜结合蛋白质或蛋白质-蛋白质复合物的功能形式，也将使结构测定在高分辨率的NMR光谱。我们选择细胞色素蛋白（CytP 450、CytP 450-还原酶（CYPOR）和Cytb 5）及其复合物（P450-CYPOR和P450- b5）作为优化的模型系统。这些膜蛋白所带来的挑战是多方面的，因此，为解决这些挑战而开发的生物化学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，我们建议调查新类别的温和的洗涤剂和非洗涤剂表面活性剂，以及天然膜样bicelles，显示承诺在稳定的天然折叠膜蛋白质使用快速分配免费筛选过程通过NMR光谱。虽然这项建议的重点是对NMR结构研究的样品条件的快速优化，但其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）的调查。 公共卫生相关性：关于膜蛋白的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，并将有助于开发治疗各种疾病的药物。

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.