Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio 45056, USA.
J Am Chem Soc. 2010 Sep 1;132(34):11910-3. doi: 10.1021/ja105080h.
There is a general need to develop more powerful and more robust methods for structural characterization of homodimers, homo-oligomers, and multiprotein complexes using solution-state NMR methods. In recent years, there has been increasing emphasis on integrating distinct and complementary methodologies for structure determination of multiprotein complexes. One approach not yet widely used is to obtain intermediate and long-range distance constraints from paramagnetic relaxation enhancements (PRE) and electron paramagnetic resonance (EPR)-based techniques such as double electron electron resonance (DEER), which, when used together, can provide supplemental distance constraints spanning to 10-70 A. In this Communication, we describe integration of PRE and DEER data with conventional solution-state nuclear magnetic resonance (NMR) methods for structure determination of Dsy0195, a homodimer (62 amino acids per monomer) from Desulfitobacterium hafniense. Our results indicate that combination of conventional NMR restraints with only one or a few DEER distance constraints and a small number of PRE constraints is sufficient for the automatic NMR-based structure determination program CYANA to build a network of interchain nuclear Overhauser effect constraints that can be used to accurately define both the homodimer interface and the global homodimer structure. The use of DEER distances as a source of supplemental constraints as described here has virtually no upper molecular weight limit, and utilization of the PRE constraints is limited only by the ability to make accurate assignments of the protein amide proton and nitrogen chemical shifts.
目前，人们普遍需要开发更强大、更稳健的方法，以便使用溶液状态 NMR 方法对同二聚体、同寡聚体和多蛋白复合物进行结构特征分析。近年来，人们越来越重视整合不同的、互补的方法，以确定多蛋白复合物的结构。一种尚未广泛应用的方法是从顺磁弛豫增强（PRE）和基于电子顺磁共振（EPR）的技术（如双电子电子共振（DEER））中获得中间和长程距离约束，这些方法结合使用可以提供补充的距离约束，跨度为 10-70Å。在本通讯中，我们描述了将 PRE 和 DEER 数据与传统的溶液状态核磁共振（NMR）方法相结合，用于确定来自脱硫脱硫弧菌的同二聚体（每个单体 62 个氨基酸）Dsy0195 的结构。我们的结果表明，将常规 NMR 约束与仅一个或几个 DEER 距离约束和少量 PRE 约束相结合，足以使基于 NMR 的自动结构确定程序 CYANA 构建一个链间核 Overhauser 效应约束网络，该网络可用于准确定义同二聚体界面和全局同二聚体结构。如这里所述，将 DEER 距离用作补充约束的来源实际上没有分子量上限，并且 PRE 约束的使用仅受准确分配蛋白质酰胺质子和氮化学位移的能力限制。
