Rapid and efficient production of functional Cytochrome P450 enzymatic complex fo

描述(申请人提供)：膜蛋白是许多细胞和生理过程的基本调节器，包括细胞之间的信号传递，跨细胞膜的运输和能量传递过程。膜蛋白的高分辨率结构可以提供对其功能的洞察，并使化合物的开发能够改变其生物和生物医学目的的性质。虽然超过30%的人类基因组和50%的已知药物靶标是膜蛋白，但很少有人知道膜蛋白的结构。对于大多数膜蛋白来说，这是由于缺乏膜模拟物，使蛋白质处于稳定、功能和均一的状态。在这一应用中，我们建议开发稳健的方法来生产近天然的模型膜，既能稳定膜结合的蛋白质或蛋白质-蛋白质复合体的功能形式，又能通过核磁共振光谱在高分辨率下确定结构。我们选择了细胞色素蛋白(CytP450、CytP450-还原酶和Cytb5)及其络合物(P450-CYPOR和P450-b5)作为模型体系进行优化。这些膜蛋白带来的挑战是多方面的，因此，为应对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，我们建议研究新类型的温和洗涤剂和非洗涤剂表面活性剂以及天然膜状双分子，它们通过核磁共振光谱快速免分配筛选过程显示出在稳定膜蛋白天然折叠方面的前景。虽然这项建议的重点是快速优化核磁共振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进行调查，包括EPR、FRET和X射线结晶学。 与公众健康相关：关于膜蛋白的生产和优化的拟议研究结果将大大提高我们在原子水平上获得结构和功能洞察的能力，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.