Turnbull W Bruce, Precious Bernie L, Homans Steve W
Astbury Centre for Structural Molecular Biology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
J Am Chem Soc. 2004 Feb 4;126(4):1047-54. doi: 10.1021/ja0378207.
The complex of cholera toxin and ganglioside GM1 is one of the highest affinity protein-carbohydrate interactions known. Herein, the GM1 pentasaccharide is dissected into smaller fragments to determine the contribution of each of the key monosaccharide residues to the overall binding affinity. Displacement isothermal titration calorimetry (ITC) has allowed the measurement of all of the key thermodynamic parameters for even the lowest affinity fragment ligands. Analysis of the standard free energy changes using Jencks' concept of intrinsic free energies reveals that the terminal galactose and sialic acid residues contribute 54% and 44% of the intrinsic binding energy, respectively, despite the latter ligand having little appreciable affinity for the toxin. This analysis also provides an estimate of 25.8 kJ mol(-1) for the loss of independent translational and rotational degrees of freedom on complexation and presents evidence for an alternative binding mode for ganglioside GM2. The high affinity and selectivity of the GM1-cholera toxin interaction originates principally from the conformational preorganization of the branched pentasaccharide rather than through the effect of cooperativity, which is also reinvestigated by ITC.
霍乱毒素与神经节苷脂GM1的复合物是已知的亲和力最高的蛋白质 - 碳水化合物相互作用之一。在此，将GM1五糖分解为更小的片段，以确定每个关键单糖残基对整体结合亲和力的贡献。置换等温滴定量热法（ITC）甚至可以测量最低亲和力片段配体的所有关键热力学参数。使用詹克斯的内在自由能概念对标准自由能变化进行分析表明，尽管后者配体对毒素的亲和力很小，但末端半乳糖和唾液酸残基分别贡献了54%和44%的内在结合能。该分析还估计了络合时独立平移和旋转自由度损失为25.8 kJ mol(-1)，并为神经节苷脂GM2的另一种结合模式提供了证据。GM1 - 霍乱毒素相互作用的高亲和力和选择性主要源于分支五糖的构象预组织，而非协同效应，ITC也对协同效应进行了重新研究。
