Department of Bioengineering Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
ACS Chem Neurosci. 2010 Jul 21;1(7):495-504. doi: 10.1021/cn1000205. Epub 2010 Apr 9.
Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach.
在生物学中，大分子识别小分子扩散性分子是普遍存在的。为了研究这些相互作用，将小分子配体固定在基底上是很重要的；然而，在这种情况下，保持与生物分子结合伙伴的识别是具有挑战性的。我们已经开发了用血清素修饰基底的方法，血清素是一种在大脑功能和精神疾病中起重要作用的小分子神经递质。为了模拟可溶性血清素，我们通过辅助羧酸基团将其氨基酸前体 5-羟色氨酸连接到寡聚（乙二醇）末端烷硫醇自组装在金上。抗 5-羟色氨酸抗体识别这些底物，证明了其生物可用性。有趣的是，5-羟色氨酸功能化表面可以对膜相关的血清素受体进行对映体特异性捕获。相比之下，用血清素本身功能化的表面不能结合血清素受体。我们推断，生物分子的识别是为了区分溶液中的小分子配体，这需要通过异位部分来连接后者。使用这种可推广的方法，可以捕获膜蛋白（众所周知，膜蛋白很难分离）或其他结合伙伴，用于鉴定、映射、表达和其他目的。
