Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein
Fatty acid interactions with a helix-less variant of intestinal fatty acid-binding protein
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DOI：
10.1021/bi952912x
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发表时间：
1996-06-11
期刊：
影响因子：
2.9
通讯作者：
Frieden, C
中科院分区：
文献类型：
--
作者：
Cistola, DP;Kim, K;Frieden, C
肠脂肪酸结合蛋白（I-FABP）在由两个反向平行的β折叠包围的封闭腔中结合单个长链脂肪酸分子。该结构还含有两个短的α-螺旋，其在与脂肪酸的甲基末端相邻的结合腔的一端上形成帽。在这项研究中，我们使用了称为Delta 17-SG的I-FABP的无螺旋变体[Kim，K.，Cistola，D. P.，& Frieden，C.（1996）Biochemistry 35，7553-7558]，以研究螺旋区在维持结合腔的完整性和介导配体的获得中的作用。荧光和NMR实验被用来表征能量，结构和动力学特性的脂肪酸结合到这个变体，并与野生型I-FABP和单位点突变体，R106 T的结果进行了比较和对比。值得注意的是，油酸结合Delta 17-SG的解离常数为4.5 μ M，该值与R106 T相当，比野生型I-FABP高约20-100倍。与Delta 17-SG复合的[2-C-13]棕榈酸酯的异源二维NMR光谱显示了与野生型蛋白质观察到的几乎相同的模式，但与R106 T不同。此外，结合的[1-C-13]棕榈酸酯的电离行为和来自C-13过滤的NOESY实验的[2-C-13]棕榈酸酯的最近邻模式对于Delta 17-SG和野生型蛋白非常相似。这些结果表明，在野生型蛋白质中的脂肪酸结合腔的羧基端的脂肪酸-蛋白质相互作用特征在无螺旋变体中基本上是完整的。相反，与Delta 17-SG结合的[16-C-13]棕榈酸酯的C-13过滤NOESY光谱表明，结合腔甲基端的脂肪酸-蛋白质相互作用被破坏。通过停流荧光法测定，Delta 17-SG和野生型I-FABP的配体结合速率均随油酸盐浓度的增加而增加，但只有野生型蛋白表现出1000 s（-1）的极限值。这一限速过程被解释为涉及螺旋区域的构象变化，其允许配体进入内腔。动力学结果的模拟和拟合得到Delta 17-SG和野生型I-FABP的配体缔合速率相当。然而，野生型蛋白的解离速率比Delta 17-SG低16倍。我们的结论是，α-螺旋的I-FABP是不需要保持完整的脂肪酸结合腔，但可以用来调节的亲和力，通过选择性地改变解离速率常数的脂肪酸结合。以这种方式，涉及α-螺旋结构域的构象变化可能有助于控制脂肪酸在细胞内的转移。
Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two antiparallel beta-sheets. The structure also contains two short alpha-helices which form a cap over one end of the binding cavity adjacent to the methyl terminus of the fatty acid. In this study, we employed a helix-less variant of I-FABP known as Delta 17-SG [Kim, K., Cistola, D. P., & Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and kinetic properties of fatty acid binding to this variant, and the results were compared and contrasted with those of wild-type I-FABP and a single-site mutant, R106T. Remarkably, oleate bound to Delta 17-SG with a dissociation constant of 4.5 mu M, a value comparable to that for R106T and approximately 20-100-fold higher than that for wild-type I-FABP. Heteronuclear two-dimensional NMR spectra for [2-C-13]palmitate complexed with Delta 17-SG revealed a pattern nearly identical to that observed for the wild-type protein, but distinct from that for R106T. In addition, the ionization behavior of bound [1-C-13]palmitate and the nearest neighbor patterns for [2-C-13]palmitate derived from C-13-filtered NOESY experiments were very similar for Delta 17-SG and the wild-type protein. These results implied that the fatty acid-protein interactions characteristic of the carboxyl end of the fatty acid binding cavity in the wild-type protein were essentially intact in the helix-less variant. In contrast, C-13-filtered NOESY spectra of [16-C-13]palmitate bound to Delta 17-SG indicated that the fatty acid-protein interactions at the methyl end of the binding cavity were disrupted. As determined by stopped-flow fluorescence, the observed ligand association rates for both Delta 17-SG and wild-type I-FABP increased with increasing oleate concentration, but only the wild-type protein exhibited a limiting value of 1000 s(-1). This rate-limiting process was interpreted as a conformational change involving the helical region that allows the ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for Delta 17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for Delta 17-SG. We conclude that the alpha-helices of I-FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regulate the affinity of fatty acid binding by selectively altering the dissociation rate constant. In this manner, conformational changes involving the alpha-helical domain may help control the transfer of fatty acids within the cell.