Integrated Theoretical and Experimental Studies of P450 Structure and Function

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将膜蛋白封装在可溶性纳米盘结构中的新方法结合起来，并通过固态核磁共振（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）来试验这些方法，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起关键作用。这些膜结合蛋白的表达利用实验纳米盘系统，允许膜蛋白在稳定的脂蛋白复合物中以活性的天然构象存在，为固态核磁共振分析提供单分散蛋白。使用高场（600-750 MHz 1H频率）魔角旋转核磁共振波谱和多维偶极重耦合脉冲序列，将为普遍存在的和困难的一类膜蛋白提供低分辨率的结构骨干信息，这些蛋白质不易被许多用于可溶性蛋白质的技术在结构水平上表征。我们将以完全跨学科的方式，利用我们的分子建模能力来促进SSNMR光谱的分辨率和我们的SSNMR衍生的骨干结构，以限制需要分析和侧链取向结构预测的分子模型的范围。分子建模和SSNMR之间的界面自动化具有显著的潜力，可以提高对多种蛋白质结构的定义能力，不仅包括膜结合的P450蛋白质，还包括能够在原核生物（大肠杆菌）和真核生物（杆状病毒感染的昆虫细胞，酵母）表达系统中表达的所有蛋白质。对这个由不同P450序列组成的非常大的超家族中的蛋白质进行结构比较，将使我们能够确定当前分子建模程序的限制，对比常见代谢活动的进化不同解决方案，并开始准确预测P450的结构，而P450的晶体结构可能永远不可能实现。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，并确定其截短形式和可溶性形式衍生的可用晶体结构的差异。

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.