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: 7862634
• 负责人: Mary A Schuler
• 金额: $ 53.56万
• 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 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7862634
• 关键词: Active Sites; Automation; Baculoviruses; Binding; Binding Proteins; Biochemistry; Cells; Chemicals; Chemistry; 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; Molecular Models; N-terminal; NMR Spectroscopy; Physiologic pulse; Pilot Projects; Plants; Play; Procedures; Process; Proteins; 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; protein complex; research study; solid state nuclear magnetic resonance; 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用于将膜蛋白封装在可溶性Nanodisc结构中的新方法相结合，并通过固态NMR（SSNMR）获得精确的三维结构，以获得膜蛋白的准确预测。拟议的项目通过分析有限数量的细胞色素P450单加氧酶（P450）来试点这些方法，这些酶在哺乳动物，昆虫，植物，真菌和细菌系统的合成和解毒功能中发挥关键作用。这些膜结合蛋白的表达利用实验Nanodisc系统，该系统允许膜蛋白以稳定的脂质-蛋白质复合物内的活性天然构象存在，以提供用于固态NMR分析的单分散蛋白质。使用高场（600-750 MHz 1H频率）魔角旋转NMR光谱和多维偶极再耦合脉冲序列将提供低分辨率的结构骨干信息的普遍存在的和困难的一类膜蛋白，不容易在结构水平上的特点，许多技术应用于可溶性蛋白质。我们将以一种完全跨学科的方式，利用我们的分子建模能力来促进SSNMR光谱和我们的SSNMR衍生骨架结构的分辨率，以限制需要分析和侧链方向结构预测的分子模型的范围。分子建模和SSNMR之间接口的自动化具有显著的潜力，可以提高定义广泛蛋白质结构的能力，不仅是膜结合的P450蛋白，而且是能够在原核表达的整个蛋白质范围（E.大肠杆菌）和真核（杆状病毒感染的昆虫细胞，酵母）表达系统。在这个非常大的不同P450序列的超家族内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，将进化的不同解决方案与常见的代谢活动进行对比，并开始准确地预测P450的结构，而晶体结构可能永远不会成为可能。这些研究还将允许在脂质相关环境中表征这些蛋白质，该环境应概括这些蛋白质与其天然膜的相互作用，并确定与其截短和可溶形式衍生的可用晶体结构的差异。