Zhuang Tiandi, Leffler Hakon, Prestegard James H
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA.
Protein Sci. 2006 Jul;15(7):1780-90. doi: 10.1110/ps.051994306. Epub 2006 Jun 2.
Residual dipolar couplings (RDCs) have proven to be a valuable NMR tool that can provide long-range constraints for molecular structure determination. The constraints are orientational in nature and are, thus, highly complementary to conventional distance constraints from NOE data. This complementarity would seem to extend to the study of the geometry of ligands bound to proteins. However, unlike transferred NOEs, where collection, even with a large excess of free ligand, results in measurements dominated by bound contributions, RDCs of exchanging ligands can be dominated by free-state contributions. Here we present a strategy for enhancement of RDCs from bound states that is based on specifically enhancing the alignment of the protein to which a ligand will bind. The protein is modified by addition of a hydrophobic alkyl tail that anchors it to the bicelles that are a part of the ordering medium needed for RDC measurement. As an illustration, we have added a propyl chain to the C terminus of the carbohydrate recognition domain of the protein, Galectin-3, and report enhanced RDCs that prove consistent with known bound-ligand geometries for this protein.
剩余偶极耦合（RDCs）已被证明是一种有价值的核磁共振工具，可用于为分子结构测定提供远程约束。这些约束本质上是取向性的，因此与来自NOE数据的传统距离约束具有高度互补性。这种互补性似乎也适用于对与蛋白质结合的配体几何结构的研究。然而，与转移NOE不同，即使存在大量过量的游离配体，收集转移NOE时，测量结果仍以结合态贡献为主，而交换配体的RDCs可能以游离态贡献为主。本文我们提出了一种增强结合态RDCs的策略，该策略基于特异性增强配体将要结合的蛋白质的排列。通过添加疏水烷基尾对蛋白质进行修饰，该烷基尾将蛋白质锚定到双分子层上，双分子层是RDC测量所需有序介质的一部分。作为一个例子，我们在蛋白质半乳糖凝集素-3（Galectin-3）的碳水化合物识别结构域的C末端添加了一个丙基链，并报告了增强的RDCs，这些结果证明与该蛋白质已知的结合配体几何结构一致。
