Marsden B J, Shaw G S, Sykes B D
Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont.
Biochem Cell Biol. 1990 Mar;68(3):587-601. doi: 10.1139/o90-084.
This paper describes the sequence homology of calcium-binding proteins belonging to the troponin C superfamily. Specifically, this similarity has been examined for 276 twelve-residue calcium-binding loops. It has been found that, in the calcium-binding loop, several residues appear invariant, regardless of the species of origin or the affinity of the protein. These residues are Asp at position 1 (+X of the coordinating position of the calcium), Asp or Asn at position 3 (+Y), Gly at position 6, Ile at position 8, and Glu at position 12 (-Z). It has also been found that conservation of certain residues can vary in similar sites in similar proteins. For example, position 3 (+Y) in site 3 of troponin C is always an Asn, whereas in calmodulin the residue is always Asp. This study also examined the calcium-binding affinities of peptide fragments comprising the loop, helix-loop, loop-helix, and helix-loop-helix. These were compared with larger enzymatic or chemically generated protein fragments in an effort to understand the various contributions to the calcium-binding affinity of a single-site versus a two-site domain as found in troponin C and calmodulin. Based on free energy differences, it was found that a 34-residue helix-loop-helix peptide represents about 60% of the binding affinity found in the intact protein. Cooperativity with a second calcium binding site accounted for the remaining 40% of the affinity.
本文描述了肌钙蛋白C超家族中钙结合蛋白的序列同源性。具体而言，已对276个十二残基的钙结合环进行了这种相似性研究。研究发现，在钙结合环中，无论蛋白质的来源物种或亲和力如何，有几个残基似乎是不变的。这些残基分别是第1位的天冬氨酸（钙配位位置的+X）、第3位的天冬氨酸或天冬酰胺（+Y）、第6位的甘氨酸、第8位的异亮氨酸以及第12位的谷氨酸（-Z）。还发现某些残基的保守性在相似蛋白质的相似位点可能会有所不同。例如，肌钙蛋白C第3位点的第3位（+Y）总是天冬酰胺，而在钙调蛋白中该残基总是天冬氨酸。本研究还检测了包含环、螺旋-环、环-螺旋和螺旋-环-螺旋的肽片段的钙结合亲和力。将这些与更大的酶促或化学生成的蛋白质片段进行比较，以试图了解如在肌钙蛋白C和钙调蛋白中发现的单一位点与双位点结构域对钙结合亲和力的各种贡献。基于自由能差异，发现一个34残基的螺旋-环-螺旋肽代表了完整蛋白质中约60%的结合亲和力。与第二个钙结合位点的协同作用占剩余40%的亲和力。
