Hahn K M, Waggoner A S, Taylor D L
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
J Biol Chem. 1990 Nov 25;265(33):20335-45.
Structure-activity studies of tetramethinemerocyanine fluorophores enabled the synthesis of novel dyes which showed spectral changes during reversible, calcium-dependent association with calmodulin. These spectral changes were greatly enhanced in dyes with a quaternary nitrogen and specifically placed hydrophobic chains. One such dye was covalently attached to calmodulin, producing a calmodulin analog with calcium-sensitive fluorescence. The analog, MeroCaM, showed a calcium-induced 3.4-fold increase in excitation ratio (608/532 nm excitation, 623 nm emission), which was fully reversed by lowering free calcium levels. MeroCaM's excitation ratio showed a half-maximal change at 300-400 nM calcium, below calcium concentrations reported to produce half-maximal saturation of calcium-calmodulin binding. However, the calcium dependence of MeroCaM's phosphodiesterase activation paralleled that of calmodulin. MeroCaM's fluorescence changes therefore appear to reflect primarily calcium binding to high affinity sites. MeroCaM's maximal phosphodiesterase activation was 30-40% that of calmodulin. In myosin light chain kinase activation, MeroCaM and calmodulin displayed indistinguishable maximal activation levels and concentration dependence of activation. Changes in MeroCaM's calcium affinity induced by magnesium, phosphodiesterase, and melittin were similar to those reported for calmodulin. Experiments with melittin revealed that target protein interaction could alter the fluorescence changes produced by calcium binding. MeroCaM showed promising brightness and photostability when imaged in individual living fibroblasts. The long excitation and emission wavelengths of MeroCaM, and the strong dependence of its excitation ratio on calcium concentrations, suit it well for use as a probe of calmodulin-dependent calcium signaling in living cells, as well as for experiments in vitro.
对四甲川部花青荧光团进行的构效关系研究促成了新型染料的合成，这些染料在与钙调蛋白进行可逆的、钙依赖性结合过程中呈现出光谱变化。在带有季铵氮和特定位置疏水链的染料中，这些光谱变化得到了极大增强。其中一种染料与钙调蛋白共价连接，产生了一种具有钙敏荧光的钙调蛋白类似物。这种类似物，即MeroCaM，在激发比（608/532 nm激发，623 nm发射）上显示出钙诱导的3.4倍增加，通过降低游离钙水平可使其完全逆转。MeroCaM的激发比在300 - 400 nM钙浓度时出现半数最大变化，该钙浓度低于据报道产生钙 - 钙调蛋白结合半数最大饱和度的浓度。然而，MeroCaM对磷酸二酯酶激活的钙依赖性与钙调蛋白的情况相似。因此，MeroCaM的荧光变化似乎主要反映了钙与高亲和力位点的结合。MeroCaM对磷酸二酯酶的最大激活程度为钙调蛋白的30 - 40%。在肌球蛋白轻链激酶激活过程中，MeroCaM和钙调蛋白表现出难以区分的最大激活水平和激活浓度依赖性。镁、磷酸二酯酶和蜂毒素诱导的MeroCaM钙亲和力变化与报道的钙调蛋白的变化相似。用蜂毒素进行的实验表明，靶蛋白相互作用可改变钙结合产生的荧光变化。当在单个活的成纤维细胞中成像时，MeroCaM显示出良好的亮度和光稳定性。MeroCaM的长激发和发射波长，以及其激发比对钙浓度的强烈依赖性，使其非常适合用作活细胞中钙调蛋白依赖性钙信号传导的探针，以及用于体外实验。
