Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase.
Conformational transition accompanying the binding of Ca2+ to the protein activator of 3',5'-cyclic adenosine monophosphate phosphodiesterase.
复制标题
伴随 Ca2 与 3,5-环单磷酸腺苷磷酸二酯酶的蛋白质激活剂结合而发生的构象转变。
作者：
C. Klee
3‘：5’-环磷酸腺苷二酯酶的钙依赖蛋白激活剂与2摩尔的钙离子/激活剂结合后发生构象转变。圆二色谱研究表明，Ca~(2+)诱导α-螺旋含量增加5-8%，随之而来的是无规卷曲的数量减少。在无Ca~(2+)和[乙撑双(氧乙烯硝基)]四乙酸乙酯(EGTA)存在的情况下，该蛋白含有30-35%的α螺旋、50%的无规卷曲和15-20%的β-折叠鞘。分光光度滴定表明，这两个酪氨酸残基的pk分别为10.4和11.9，因此处于不同的环境中。钙离子诱导的构象变化伴随着部分暴露的酪氨酸质子暴露的增加，如其pk从10.4到10。溶剂化增强也与钙离子结合所见的287和279 nm处的负差分光谱相一致。所有或部分苯丙氨酸残基的环境修饰也是伴随着钙结合的构象变化的一部分。本文还介绍了一种新的、快速的纯化方法和cAMP磷酸二酯酶及其激活剂的直接、灵敏的测定方法，该方法可获得大量(产率25-30%)的均一蛋白激活剂。
The Ca2+-dependent protein activator of 3':5'-cyclic adenosine monophosphate phosphodiesterase is shown to undergo a conformational transition upon binding of 2 mol of Ca2+/mol of activator. Circular dichroic studies indicate that Ca2+ induces an increase of 5-8% in alpha-helix content with a concomitant decrease in the amount of random coil. In the absence of Ca2+ and in the presence of [ethylenebis(oxoethylenenitrilo)]tetraacetic acid (EGTA), the protein contains 30-35% alpha helix, 50% random coil, and 15-20% beta-pleated sheat. Spectrophotometric titration indicates that the two tyrosyl residues have pK's of 10.4 and 11.9 and are therefore in different environments. The Ca2+-induced conformational change is accompanied by an increased exposure to protons of the partially exposed tyrosine, as shown by a shift in its pK from 10.4 to 10.). Increased solvation is also consistent with a negative difference spectrum at 287 and 279 nm as seen upon Ca2+ binding. Modification in the environment of all or some of the phenylalanine residues also is part of the conformational change accompanying Ca2+ binding. A new and rapid purification procedure which yields large amounts (25-30% yields) of homogenous protein activator and a direct and sensitive assay procedure for cAMP phosphodiesterase and its activator are also described.