Hing A W, Tjandra N, Cottam P F, Schaefer J, Ho C
Department of Chemistry, Washington University, St. Louis, Missouri 63130.
Biochemistry. 1994 Jul 26;33(29):8651-61. doi: 10.1021/bi00195a005.
Glutamine-binding protein (GlnBP) is an essential component of the glutamine transport system in Escherichia coli. Rotational-echo double-resonance (REDOR) solid-state nuclear magnetic resonance (NMR) has been used to determine internuclear distances in the complex of GlnBP and its ligand, L-glutamine. REDOR, combined with strategically placed isotopic labels, is effective in obtaining model-independent internuclear distances and thus detailed structural information on the ligand-binding site of GlnBP. The existence of a single histidine residue (His156) in the binding site has provided an excellent probe for distance measurements between protein and ligand. REDOR distances up to 6.3 A have been observed between 13C labels in L-glutamine and 15N labels in His156. These results have unambiguously determined the ligand orientation with respect to the imidazole ring of His156, which is an important first step in refining the ligand-binding-site model of GlnBP in general. The measured distances were also used as constraints in restrained molecular dynamics calculations of the complex using the unliganded crystal structure of GlnBP as the starting point. The simulations clearly show consistency between calculated distances and those measured by REDOR.
谷氨酰胺结合蛋白（GlnBP）是大肠杆菌谷氨酰胺转运系统的重要组成部分。旋转回波双共振（REDOR）固态核磁共振（NMR）已被用于确定GlnBP与其配体L-谷氨酰胺复合物中的核间距离。REDOR与精心放置的同位素标记相结合，能有效地获得与模型无关的核间距离，从而获得关于GlnBP配体结合位点的详细结构信息。结合位点中单个组氨酸残基（His156）的存在为蛋白质与配体之间的距离测量提供了一个绝佳的探针。在L-谷氨酰胺中的13C标记与His156中的15N标记之间观察到了高达6.3埃的REDOR距离。这些结果明确地确定了配体相对于His156咪唑环的取向，这总体上是完善GlnBP配体结合位点模型的重要第一步。所测量的距离也被用作以GlnBP的无配体晶体结构为起点对复合物进行受限分子动力学计算的约束条件。模拟结果清楚地表明计算距离与REDOR测量距离之间具有一致性。
