Zhang F, Lücke C, Baier L J, Sacchettini J C, Hamilton J A
Department of Biophysics, Boston University School of Medicine, Boston, MA 02118, USA.
J Biomol NMR. 1997 Apr;9(3):213-28. doi: 10.1023/a:1018666522787.
The human intestinal fatty acid binding protein (I-FABP) is a small (131 amino acids) protein which binds dietary long-chain fatty acids in the cytosol of enterocytes. Recently, an alanine to threonine substitution at position 54 in I-FABP has been identified which affects fatty acid binding and transport, and is associated with the development of insulin resistance in several populations including Mexican-Americans and Pima Indians. To investigate the molecular basis of the binding properties of I-FABP, the 3D solution structure of the more common form of human I-FABP (Ala54) was studied by multidimensional NMR spectroscopy. Recombinant I-FABP was expressed from E. coli in the presence and absence of 15N-enriched media. The sequential assignments for non-delipidated I-FABP were completed by using 2D homonuclear spectra (COSY, TOCSY and NOESY) and 3D heteronuclear spectra (NOESY-HMQC and TOCSY-HMQC). The tertiary structure of human I-FABP was calculated by using the distance geometry program DIANA based on 2519 distance constraints obtained from the NMR data. Subsequent energy minimization was carried out by using the program SYBYL in the presence of distance constraints. The conformation of human I-FABP consists of 10 antiparallel beta-strands which form two nearly orthogonal beta-sheets of five strands each, and two short alpha-helices that connect the beta-strands A and B. The interior of the protein consists of a water-filled cavity between the two beta-sheets. The NMR solution structure of human I-FABP is similar to the crystal structure of rat I-FABP. The NMR results show significant conformational variability of certain backbone segments around the postulated portal region for the entry and exit of fatty acid ligand.
人肠脂肪酸结合蛋白（I-FABP）是一种小蛋白（131个氨基酸），在肠细胞的胞质溶胶中结合膳食长链脂肪酸。最近，已鉴定出I-FABP第54位的丙氨酸被苏氨酸取代，这会影响脂肪酸的结合和转运，并与包括墨西哥裔美国人及皮马印第安人在内的多个群体中胰岛素抵抗的发展有关。为了研究I-FABP结合特性的分子基础，通过多维核磁共振光谱研究了人I-FABP更常见形式（Ala54）的三维溶液结构。在有和没有15N富集培养基的情况下，从大肠杆菌中表达重组I-FABP。通过使用二维同核光谱（COSY、TOCSY和NOESY）和三维异核光谱（NOESY-HMQC和TOCSY-HMQC）完成了非脱脂I-FABP的序列归属。基于从核磁共振数据获得的2519个距离约束，使用距离几何程序DIANA计算人I-FABP的三级结构。随后在距离约束存在的情况下，使用SYBYL程序进行能量最小化。人I-FABP的构象由10条反平行β链组成，这些β链形成两个几乎正交的β折叠片层，每个片层有五条链，以及连接β链A和B的两个短α螺旋。蛋白质内部由两个β折叠片层之间的一个充满水的腔组成。人I-FABP的核磁共振溶液结构与大鼠I-FABP的晶体结构相似。核磁共振结果表明，在假定的脂肪酸配体进出的门户区域周围，某些主链片段存在显著的构象变异性。
