Linse S, Chazin W J
Department of Physical Chemistry 2, University of Lund, Sweden.
Protein Sci. 1995 Jun;4(6):1038-44. doi: 10.1002/pro.5560040602.
Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k.
正协同效应是指由于在另一个位点结合相同类型的配体而导致一个位点的配体亲和力增强，它是结合位点之间的一种自由能偶联。它既可以存在于具有相同配体亲和力位点的系统中，也可以存在于结合位点具有不同亲和力的系统中。当这些位点的亲和力差异很大，以至于它们以相继的方式被配体占据时，如果存在正协同效应，通常很难对其进行量化甚至检测。本研究展示了对钙结合蛋白D9k突变体形式中两个钙结合位点之间自由能偶联的验证和定量测量。野生型钙结合蛋白D9k以相似的亲和力和正协同效应结合两个钙离子——自由能偶联ΔΔG约为 -8 kJ·mol⁻¹（林斯·S等人，1991年，《生物化学》30卷：154 - 162页）。该突变体将天冬酰胺56替换为丙氨酸，以相继的方式结合钙。在本研究中，我们利用了不同金属离子对钙结合蛋白D9k中两个结合位点的偏好差异。对Ca²⁺、Cd²⁺和La³⁺结合的联合研究使我们得出结论，在这个突变体中ΔΔG < -6.4 kJ·mol⁻¹，并且Cd²⁺和La³⁺也以正协同效应结合到该蛋白质上。这些结果证明将天冬酰胺56替换为丙氨酸突变体的（Ca²⁺）¹状态以及野生型的（Cd²⁺）¹状态用作钙结合蛋白D9k中协同Ca²⁺结合两条途径上半饱和状态模型的合理性。
