Molecular reconstruction of flavocytochrome P450 BM3

确定生物分子的结构对于理解其生理功能至关重要。优秀的例子包括克里克和沃森的DNA结构，马克斯·佩鲁茨解决的血红蛋白结构以及（最近）复杂分子机器（ATP合成酶和呼吸复合物）的结构，这些结构有助于从食物（脂质，碳水化合物等）的氧化中产生能量，从而使生命得以存在。用于确定生物（和其他）分子中原子的三维组织的主要技术是X射线晶体学（来自分子晶体的X射线衍射，例如蛋白质）和用于溶液中分子的核磁共振（NMR）。然而，这些方法用于大生物分子（如复杂蛋白质和酶）的结构测定存在局限性。通常，确定尺寸大于30 kDa的分子的结构对于NMR来说变得具有挑战性和昂贵。虽然X射线衍射方法对所讨论的分子的大小更“宽容”，但它绝对依赖于获得目标分子晶体的能力。这可能是困难的（或不可能的），即使获得了晶体，它们也可能无法很好地吸收X射线，从而无法解决结构问题。含有几个亚基或单个结构片段（结构域）的大型复杂蛋白质的晶体通常难以结晶，特别是如果蛋白质的区域在溶液中高度移动的。P450在人类药物/异生物质解毒中至关重要，并且在类固醇代谢中起重要作用。它们通过在基质中引入氧原子来实现这一点。P450由于其在药物代谢中的重要性而被广泛研究，并且本身是重要的药物靶点，但迄今为止还没有真正的结构数据来描述P450如何与其伴侣酶-特别是细胞色素P450还原酶（CPR）相互作用。在这项工作中，我们将应用一种新的工具组合（光谱，建模，蛋白质工程和分析方法）来定义BM 3酶的结构组织-这是P450酶家族中的“模型”系统，也是一种天然的“融合酶”，由脂肪酸代谢P450直接连接到CPR酶形成。虽然哺乳动物P450和CPR是膜相关的不溶性蛋白质，但BM 3是完全可溶的，并且可以大量制备用于表征。BM 3蛋白有几个结构域，我们最近已经表明，它是作为一个二聚体的功能。BM 3可以被分解（通过基因工程）成其单个结构域（例如CPR和P450模块），并且其单个结构域的结构已经通过X射线晶体学解析。然而，由于上述原因，完整的BM 3对晶体学是难处理的。该提案的关键是通过协同应用不同的光谱、建模和其他方法来提供BM 3二聚体的结构模型，以使得能够定义形状、结构域移动性和单体间距离的方面，从而允许重构BM 3二聚体的结构。然后将验证该模型，然后将为BM 3开发的工具应用于另一种重要的酶-一氧化氮合酶（NOS），其具有与BM 3相似的二聚体结构，但由于与钙结合蛋白钙调素的相互作用，其调节模式更复杂。NOS也不易结晶，但对人类神经传递、免疫功能和血压控制至关重要。总的来说，这些对BM 3/NOS的研究将为确定复杂多结构域蛋白的结构布局提供新的途径，并深入了解这些血红素结合蛋白如何与其连接的CPR伴侣相互作用。

Determining the structure of biological molecules is critical for understanding their physiological function. Excellent examples include Crick and Watson's DNA structure, the structure of hemoglobin solved by Max Perutz and (more recently) the structures of the complex molecular machines (ATP synthase and the respiratory complexes) that facilitate energy generation from oxidation of foodstuffs (lipids, carbohydrates etc) to enable life. The major techniques used to determine the three dimensional organization of atoms in biological (and other) molecules are X-ray crystallography (diffraction of X-rays from crystals of the molecule, e.g. a protein) and nuclear magnetic resonance (NMR) that is used on molecules in solution. However, there are limitations to application of these methods for structural determination of large biomolecules (such as complex proteins and enzymes). Typically, determining structures of molecules larger in size than 30 kDa becomes challenging and expensive for NMR. While X-ray diffraction methods are more "tolerant" to the size of the molecule in question, it is absolutely dependent on ability to obtain crystals of the target molecule. This can be difficult (or impossible), and even if crystals are obtained they may not diffract X-rays sufficiently well to enable a structure to be solved. Crystals of large, complex proteins containing several subunits or individual structural segments (domains) are often difficult to crystallize, particularly if regions of the protein are highly mobile in solution.The subject of this proposal is an important member of the cytochrome P450 (P450) class of enzymes named flavocytochrome P450 BM3 (BM3). P450s are critical in human drug/xenobiotic detoxification, and play important roles in steroid metabolism. They do this by introducing oxygen atoms into their substrates. P450s are widely studied due to their importance in drug metabolism, as well as being important drug targets in their own right, but to date there are no true structural data to describe how P450s interact with their partner enzymes -notably cytochrome P450 reductase (CPR). In this work, we will apply a new combination of tools (spectroscopic, modelling, protein engineering and analytical methods) to define the structural organization of the BM3 enzyme - which is a "model" system in the P450 enzyme family, and which is also a natural "fusion enzyme" formed by direct linkage of a fatty acid metabolizing P450 to a CPR enzyme. While mammalian P450s and CPR are membrane associated, insoluble proteins, BM3 is fully soluble and can be prepared in large amounts for characterization. The BM3 protein has several domains and we have shown recently that it is functional as a dimer. BM3 can be broken down (by genetic engineering) into its individual domains (e.g. the CPR and P450 modules) and structures of its individual domains have been solved by X-ray crystallography. However, intact BM3 has been refractory to crystallography, for reasons described above. The crux of this proposal is to provide a structural model for the BM3 dimer through synergistic application of diverse spectroscopic, modelling and other methods to enable aspects of shape, domain mobility and inter-monomer distances to be defined, allowing the structure of the BM3 dimer to be reconstructed. The model will then be validated, and tools developed for BM3 will then be applied to another important enzyme - nitric oxide synthase (NOS) which has a similar dimeric architecture to BM3, but a more complicated mode of regulation due to interactions with the calcium binding protein calmodulin. NOS has also been refractory to crystallization, but is critical for human neurotransmission, immune function and blood pressure control. Collectively, these studies on BM3/NOS will provide a new route to determining the structural layout of complex multidomain proteins, as well as insights into how these heme-binding proteins interact with their linked CPR partner.