Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin.

Binding of 13C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin--two complementary fragments corresponding to the two halves of albumin and one fragment corresponding to the carboxyl-terminal domain--yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). Two of these resonances were seen upon addition of 1 or 2 mol of oleic acid to fragments representing either the carboxyl-terminal half (residues 307-582) or the carboxyl-terminal domain (residues 377-582); the third resonance was seen upon addition of 1 mol of oleic acid to the fragment representing the amino-terminal half (residues 1-306). The resonance patterns for the fourth and fifth moles of oleic acid added to albumin (secondary sites) could not be duplicated by addition of more oleic acid to individual fragments. These resonance patterns were generated, however, when the two complementary fragments were mixed in equimolar proportions to form an albumin-like complex with a reconstituted middle domain. Thus, two primary fatty acid binding sites are assigned to the carboxyl-terminal domain, one primary site is assigned to the amino-terminal half, and the secondary sites are assigned to the middle domain. This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domains.

将13C富集的油酸（13C-enriched oleic acid）与牛血清白蛋白（bovine serum albumin）以及白蛋白的三大蛋白水解片段结合——其中两个互补片段分别对应白蛋白的两个结构半体，另一个片段对应羧基末端结构域（carboxyl-terminal domain）——得到了特征性的核磁共振（Nuclear Magnetic Resonance, NMR）共振谱图，其参数涵盖化学位移与相对强度，借此明确了前5摩尔油酸与该多结构域白蛋白分子的具体结合位点。向完整白蛋白中加入前3摩尔油酸时，由于油酸同时结合至三个异质性结合位点（初级结合位点，primary sites），会产生三组截然不同的NMR共振峰。向对应羧基末端半体（氨基酸残基307-582）或羧基末端结构域（氨基酸残基377-582）的蛋白片段加入1或2摩尔油酸时，可观察到其中两组共振峰；而向对应氨基末端半体（氨基酸残基1-306）的蛋白片段加入1摩尔油酸时，则会出现第三组共振峰。向白蛋白中加入第四、第五摩尔油酸时对应的共振谱图（对应次级结合位点，secondary sites），无法通过向单一蛋白片段中额外加入油酸来复刻。但当将两个互补片段以等摩尔比例混合，重构得到带有中间结构域的类白蛋白复合物时，即可重现该共振谱图。据此可将两个初级脂肪酸结合位点归属于羧基末端结构域，一个初级结合位点归属于氨基末端半体，而次级结合位点则归属于中间结构域。该结合位点分布表明，白蛋白的结合分子对称性较理论模型的预测结果更低。本研究同时证实了核磁共振技术可用于解析特定结构域内单个脂肪酸结合位点的微环境。