Integrated Theoretical and Experimental Studies of P450 Structure and Function

描述(申请人提供)：这个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，将膜蛋白包裹在可溶的纳米盘结构中，并通过固态核磁共振(SS核磁共振)获得精确的三维结构，以获得对膜蛋白的准确预测。拟议的项目通过对有限数量的细胞色素P450单加氧酶(P450)的分析来引导这些方法，这些单加氧酶在哺乳动物、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纳米盘系统，该系统允许膜蛋白以活跃的天然构象存在于稳定的脂-蛋白复合体中，为固体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、困难的一类膜蛋白提供低分辨率的结构骨架信息，而许多应用于可溶性蛋白的技术不容易在结构水平上表征这些膜蛋白。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SS核磁共振谱和我们的SS核磁共振衍生主干结构的解析，以限制需要分析和预测侧链取向的分子模型的范围。分子建模和SS核磁共振之间接口的自动化具有巨大的潜力，可以提高确定各种蛋白质结构的能力，不仅是膜结合的P450蛋白质，而且是适合于同位素标记的原核(大肠杆菌)和真核(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。对这个非常庞大的发散P450超家族中的蛋白质进行结构比较，将使我们能够定义当前分子建模程序的限制，将进化上不同的解决方案与常见的代谢活动进行对比，并开始准确预测P450的结构，对于这些结构，晶体结构可能永远不可能实现。这些研究还将允许在与脂质相关的环境中表征这些蛋白质，这种环境应该概括这些蛋白质与其天然膜的相互作用，并确定其截断和可溶形式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.