Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.
Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.
复制标题
EF-hand 蛋白与连续钙结合的协同性的定量测量。
DOI：
10.1002/pro.5560040602
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Chazin,WJ
中科院分区：
文献类型：
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作者：
Linse,S;Chazin,WJ
正协同性，定义为由于在另一位点结合相同类型的配体而在一个位点增强配体亲和力，是结合位点之间的自由能偶联。它既可以存在于具有相同配体亲和力的位点的系统中，也可以存在于结合位点具有不同亲和力的系统中。当位点具有广泛不同的亲和力使得它们以顺序方式填充有配体时，通常难以量化或甚至检测正协同性（如果发生的话）。本研究提出了验证和定量测量的两个钙结合位点之间的钙结合蛋白D9 K的突变形式的自由能耦合。野生型钙结合蛋白D9 k以相似的亲和力和正协同性结合两个钙离子-自由能耦合ΔΔG约为-8 kJ · mol−1（Linse S，et al.，1991，Biochemistry 30：154-162）。突变体Asn 56 → Ala以顺序方式结合钙。在目前的工作中，我们已经利用了不同的金属离子之间的变化，在他们的偏好钙结合蛋白D9 K的两个结合位点。对Ca 2+、Cd 2+和La 3+结合的综合研究使我们得出结论，在该突变体中ΔΔG <-6.4 kJ · mol−1，并且Cd 2+和La 3+也以正协同性与该蛋白结合。这些结果证明了Asn 56 → Ala突变体的（Ca 2+）1状态以及野生型的（Cd 2+）1状态作为钙结合蛋白D9 k中协同Ca 2+结合的两条途径的半饱和状态沿着的模型的合理性。
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 D9kbinds two calcium ions with similar affinities and positive cooperativity — the free energy coupling, ΔΔG, 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 ΔΔG <‐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+), state of the Asn 56 → Ala mutant, as well as the (Cd2+)1state of the wild type, as models for the half‐saturated states along the two pathways of cooperative Ca2+binding in calbindin D9k.