Integrated Theoretical and Experimental Studies of P450 Structure and Function

P450结构与功能的综合理论与实验研究

• 批准号: 7476550
• 负责人: Mary A Schuler
• 金额: $ 51.18万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7476550
• 关键词: Active Sites; Automation; Baculoviruses; Binding; Binding Proteins; Biochemistry; Cells; Chemicals; Chemistry; Class; Complex; Coupling; Cytochrome P450; Data; Developmental Cell Biology; Encapsulated; Environment; Escherichia coli; Faculty; Frequencies; Goals; Illinois; Insecta; Label; Lipids; Magic; Membrane; Membrane Proteins; Metabolic; Methodology; Mixed Function Oxygenases; Modeling; Molecular Conformation; N-terminal; NMR Spectroscopy; Numbers; Physiologic pulse; Pilot Projects; Plants; Play; Procedures; Process; Proteins; Pulse taking; Range; Research Institute; Resolution; Role; Schools; Science; Senior Scientist; Set protein; Side; Solutions; Structure; System; Techniques; Theoretical model; Universities; Vertebral column; Yeasts; improved; medical schools; molecular modeling; novel; research study; solid state; three dimensional structure

DESCRIPTION (provided by applicant): The overall goals of this project are to couple our molecular modeling capabilities with new methodologies for encapsulating membrane proteins in soluble Nanodisc structures and obtaining precise three-dimensional structures by solid-state NMR (SSNMR) to derive accurate predictions for membrane proteins. The proposed project pilots these approaches with an analysis of a limited number of cytochrome P450 monooxygenases (P450s) that play critical roles in synthetic and detoxicative functions in mammalian, insect, plant, fungal and bacterial systems. Expression of these membrane-bound proteins utilizes experimental Nanodisc systems that allow membrane proteins to exist in an active, native conformation within a stabile lipid-protein complex to provide monodisperse proteins for solid-state NMR analysis. Use of high-field (600-750 MHz 1H frequency) magic-angle spinning NMR spectroscopy and multidimensional dipolar recoupling pulse sequences will provide low-resolution structural backbone information for the ubiquitous and difficult class of membrane proteins that are not readily characterized at a structural level by many techniques applied to soluble proteins. We will, in a completely interdisciplinary fashion, use our molecular modeling capabilities to facilitate resolution of SSNMR spectra and our SSNMR-derived backbone structures to limit the range of molecular models requiring analysis and structural predictions for side-chain orientations. Automation of the interface between molecular modeling and SSNMR has significant potential for improving the capacity for defining structures on a wide range of proteins, not only membrane-bound P450 proteins but also the entire range of proteins capable of being expressed in prokaryotic (E. coli) and eukaryotic (baculovirus-infected insect cells, yeast) expression systems suitable for isotopic labeling. Structural comparisons of proteins within this very large superfamily of divergent P450 sequences will allow us to define the limits on current molecular modeling procedures, to contrast the evolutionary distinct solutions to common metabolic activities and to begin accurately predicting structures for P450s for which crystal structures may never be a possibility. These studies will also allow to characterize these proteins in a lipid-associated environment that should recapitulate interactions of these proteins with their native membranes and identify differences with available crystal structures derived for their truncated and soluble forms.

描述（由申请人提供）：该项目的总体目标是将我们的分子建模能力与新的方法结合起来，以封装可溶性纳米散布结构中的膜蛋白，并通过层固体NMR（SSNMR）获得精确的三维结构，从而得出膜蛋白的准确预测。提出的项目飞行员通过分析有限数量的细胞色素P450单加氧酶（P450）的方法，这些方法在哺乳动物，昆虫，植物，真菌和细菌系统中起着关键作用。这些结合膜结合蛋白的表达利用实验性纳米激体系统，使膜蛋白在稳定的脂质蛋白质复合物中存在于活性的天然构象中，以提供单分散蛋白进行固态NMR分析。使用高场（600-750 MHz 1H频率）的魔角旋转NMR光谱和多维二极化二极化脉冲脉冲序列将提供低分辨率的结构骨架信息，以使无处不在且困难的膜蛋白中无用的膜蛋白具有许多技术水平的溶液蛋白的特征，许多技术水平都可以通过许多技术来应用溶液蛋白。我们将以完全跨学科的方式使用分子建模能力来促进SSNMR光谱的分辨率和我们的SSNMR衍生的主链结构，以限制需要分析和结构性预测的分子模型范围。分子建模和SSNMR之间界面的自动化具有提高在广泛的蛋白质上定义结构的能力的重要潜力，这不仅是膜结合的p450蛋白，而且还可以在促脑脂性（E. coli）和真核植物（baculovirus tabirovirus tabirociational forect fected昆虫细胞中）表达的整个蛋白质范围。在这一非常大的p450序列的非常大的超家族中的蛋白质的结构比较将使我们能够定义当前分子建模程序的限制，以对比对常见代谢活性的进化不同的溶液进行对比，并开始准确预测P450的晶体结构的结构。这些研究还将允许在脂质相关的环境中表征这些蛋白质，这些蛋白质应概括这些蛋白质与其天然膜的相互作用，并与为其截断和可溶性形式得出的可用晶体结构确定差异。