Constantine K L, Friedrichs M S, Wittekind M, Jamil H, Chu C H, Parker R A, Goldfarb V, Mueller L, Farmer B T
Department of Macromolecular NMR, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Biochemistry. 1998 Jun 2;37(22):7965-80. doi: 10.1021/bi980203o.
Adipocyte lipid-binding protein (A-LBP) and muscle fatty acid-binding protein (M-FABP) are members of a family of small ( approximately 15 kDa) cytosolic proteins that are involved in the metabolism of fatty acids and other lipid-soluble molecules. Although highly homologous (65%) and structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by intrinsic protein dynamical properties, we have characterized the backbone and side chain dynamics of uncomplexed (apo) human A-LBP and M-FABP. Backbone dynamics were characterized by measurements of 15N T1 and T2 values and ¿1H¿-15N NOEs. These data were analyzed using model-free spectral density functions and reduced spectral density mapping. The dynamics of methyl-containing side chains were charaterized by measurements of 2H T1 and T1rho relaxation times of 13C1H22H groups. The 2H relaxation data were analyzed using the model-free approach. For A-LBP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 42 methyl groups. For M-FABP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 53 methyl groups. The intrinsic flexibilities of these two proteins are compared, with particular emphasis placed on binding pocket residues. There are a number of distinct dynamical differences among corresponding residues between the two proteins. In particular, many residues display greater backbone picosecond to nanosecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-helix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty acid binding pocket display a wide range of motional restriction in both proteins. Side chains showing distinct dynamical differences between the two proteins include those of residues 20, 29, and 51. This information provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced understanding of ligand affinity and selectivity among fatty acid-binding proteins.
脂肪细胞脂质结合蛋白（A-LBP）和肌肉脂肪酸结合蛋白（M-FABP）是一类小的（约15 kDa）胞质蛋白家族的成员，它们参与脂肪酸和其他脂溶性分子的代谢。尽管A-LBP和M-FABP高度同源（65%）且结构非常相似，但它们表现出不同的配体结合特性。由于配体结合可能受蛋白质内在动力学性质的影响，我们已对未结合（脱辅基）的人A-LBP和M-FABP的主链和侧链动力学进行了表征。通过测量15N T1和T2值以及1H-15N NOE来表征主链动力学。使用无模型光谱密度函数和简化光谱密度映射对这些数据进行分析。通过测量13C1H22H基团的2H T1和T1rho弛豫时间来表征含甲基侧链的动力学。使用无模型方法分析2H弛豫数据。对于A-LBP，获得了111个残基的15N弛豫数据和42个甲基的2H弛豫数据。对于M-FABP，获得了111个残基的15N弛豫数据和53个甲基的2H弛豫数据。比较了这两种蛋白质的内在柔韧性，特别强调了结合口袋残基。这两种蛋白质相应残基之间存在许多明显的动力学差异。特别是，相对于M-FABP，许多残基在A-LBP中表现出更大的皮秒到纳秒和/或微秒到毫秒时间尺度的主链流动性，包括F57、K58以及α-螺旋2中的大多数残基（残基28-35）。该区域动力学的变化可能在配体选择性中起作用。脂肪酸结合口袋内衬的侧链在两种蛋白质中都表现出广泛的运动限制。两种蛋白质之间表现出明显动力学差异的侧链包括残基20、29和51的侧链。这些信息为确定配体结合诱导的动力学变化提供了必要的基准，并最终可能有助于增强对脂肪酸结合蛋白中配体亲和力和选择性的理解。
