Jain N U, Venot A, Umemoto K, Leffler H, Prestegard J H
Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA.
Protein Sci. 2001 Nov;10(11):2393-400. doi: 10.1110/ps.17401.
The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.
研究了N-乙酰乳糖胺的氮氧化物自旋标记类似物与26kD大小的哺乳动物凝集素半乳糖凝集素-3的结合，以绘制这种小寡糖在蛋白质表面的结合位点。由于结合的自旋标记导致全长半乳糖凝集素-3的(15)N异核单量子相干(HSQC)谱中的交叉峰强度扰动，被定性地用于识别靠近N-乙酰乳糖胺结合位点的蛋白质残基。然后描述了一种将强度测量转换为更定量地确定离散蛋白质酰胺质子与结合的自旋标记之间距离的方案。该方案作为药物设计策略的一部分进行了讨论，在该策略中，距离映射的酰胺交叉峰化学位移的后续扰动可有效地用于筛选化合物库，以寻找其他在适合与初级配体进行化学连接的距离处与靶蛋白结合的配体。这种方法的新颖之处在于它绕过了对靶蛋白进行结构测定和共振归属的需要。
