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首页> 外文期刊>Biochemistry >A Nuclear Magnetic Resonance-Based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-Binding Proteins
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A Nuclear Magnetic Resonance-Based Structural Rationale for Contrasting Stoichiometry and Ligand Binding Site(s) in Fatty Acid-Binding Proteins

机译：基于核磁共振的结构原理与脂肪酸结合蛋白中的化学计量比和配体结合位点形成对比

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摘要

Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from othernfamily members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fattynacid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-ndimensionaln1nH-15nN nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABPnassemblies formed during stepwise ligand titration were exploited, without determining the protein-ligandncomplex structures, to yield the stoichiometries for the bound ligands, their locations within the proteinnbinding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations.nChemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostaticninteractions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the resultsncompared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP inncrystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chainndeep within the cavity and another extended structure with the hydrophobic chain facing the cavity and thencarboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that thenfirst oleate to enter the cavity occupies the internal protein site. In contrast,n1nHand 15nNchemical shift changesnsupported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the proteinncavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometrynand binding site trends for LFABPs and to put these findings into context within the larger protein family
机译：肝脂肪酸结合蛋白（LFABP）是一种14 kDa的胞质多肽，在配体结合位点的数量，结合的配体的多样性以及脂肪酸（主要是通过水扩散而不是通过膜的扩散）上与其他家族成员不同直接碰撞相互作用。在不确定蛋白质-配体-复合物结构的情况下，利用表明在逐步的配体滴定过程中形成的缓慢交换的LFABP的二维二维n1nH-15nN核磁共振信号来产生结合配体的化学计量，它们在蛋白质结合腔内的位置， n监测了野生型LFABP和R122L / S124A突变体的化学位移，其中突变体中的静电相互作用被认为是脂肪酸结合必不可少的。对于野生型LFABP，其结果与油酸结合的野生型LFABP inn晶体先前的三级结构数据和溶液中的数据相比较：有两个油酸盐，一个U形配体将长疏水链定位在腔内，另一个疏水结构面向空腔，然后羧酸酯基团靠近蛋白质表面延伸。 NMR滴定证实了先前的假设，即先油酸酯进入腔体占据了内部蛋白质位点。相反，n1nHand 15nN化学位移变化仅在蛋白质腔内的中间位置支持R122L / S124A LFABP的一个配体油酸酯。建立了基于蛋白质序列和静电学的原理，以解释LFABP的化学计量和结合位点趋势，并将这些发现纳入更大的蛋白质家族中

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