Bound-State Residual Dipolar Couplings for Rapidly Exchanging Ligands of His-Tagged Proteins

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• 作者：Ronald D. Seidel III ; Tiandi Zhuang ; James H. Prestegard
• 刊名：Journal of the American Chemical Society
• 出版年：2007
• 出版时间：April 18, 2007
• 年：2007
• 卷：129
• 期：15
• 页码：4834 - 4839
• 全文大小：99K
• 年卷期：v.129,no.15(April 18, 2007)
• ISSN：1520-5126

文摘

The study of bound-state conformations of ligands interacting with proteins is important to theunderstanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclearOverhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between boundand free states, have been used in these studies, owing to the inherent heavy weighting of bound-statedata in the averaged ligand signals. In principle, residual dipolar couplings (RDCs) provide a usefulcomplement to NOE data in that they provide orientational constraints as opposed to distance constraints,but use in ligand-binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound-state data in averaged RDCsmeasured on ligands is presented. The approach rests on association of a His-tagged protein with a nickel-chelate-carrying lipid inserted into the lipid bilayer-like alignment media used in the acquisition of RDCs.The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose,bound to the carbohydrate recognition domain of the mammalian lectin, galectin-3.