Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits.
Fluorescence lifetime and acrylamide quenching studies of the interactions between troponin subunits.
复制标题
肌钙蛋白亚基之间相互作用的荧光寿命和丙烯酰胺猝灭研究。
作者：
Leavis,PC;Gowell,E;Tao,T
Paul C. Leavis，* Elizabeth Gowell，and Terence Tao摘要：进行荧光寿命和丙烯酰胺淬灭研究以表征在不同金属离子结合条件下肌钙蛋白亚基之间的相互作用。肌钙蛋白C在Cys-98处用7 N-（碘乙酰基）-7N-（5-磺基-1-萘基）乙二胺标记。在Ca ~（2+）存在下，标记肌钙蛋白C（TnC ~*）的荧光衰减为单指数衰减，寿命r= 15.5ns，猝灭速率常数kq= 2.97 × 10 ~（-3）s ~（-1）。在不存在Ca ~（2+）的情况下，衰减可分解为一个主成分= 11.9 ns和一个次成分= 20.5 ns，相应的kq值分别为4.80 × 10 ~（？）和0.66 × 10 ~（？）M ~（？）在有Ca ~（2+）存在的情况下，当肌钙蛋白I（TnI）或肌钙蛋白T（TnT）结合时，r增加到~ 18 ns，kq降低到~ 0.8 × 10 ~（-1）s ~（-1）。脊椎动物骨骼肌收缩的调节是由Ca ~（2+）与肌钙蛋白复合物的肌钙蛋白C（TnC）1部分结合触发的，而肌钙蛋白C（TnC）1部分又是细丝的一部分。金属诱导的TnC结构变化被认为转移到其他肌钙蛋白亚基TnI和TnT，并最终转移到原肌球蛋白和肌动蛋白，从而影响肌动蛋白-肌球蛋白相互作用的变化。尽管该过程的细节尚不清楚，但调节机制可能涉及细丝中蛋白质-蛋白质相互作用的变化，这导致某些细丝组分（例如TnC和TnI）之间的更强结合（Margossian & Cohen，1973; Head &佩里，1974; Potter & German，1974;希区柯克，1975; Ohnishi等人，1975），以及其它的解离，例如Tnl和肌动蛋白（Margossian & Cohen，1973; Potter & German，1974;希区柯克，1975）。为了进一步阐明三种肌钙蛋白亚基之间的相互作用，我们使用荧光猝灭技术（Lehrer & Leavis，1978; Eftink & Ghiron，1981）来探测相互作用位点的微环境。猝灭参数的变化是可解释的构象变化和蛋白质间相互作用的变化，涉及标记的蛋白质。在这项工作中，用荧光探针1，5-IAEDANS（哈德逊& Weber，1973）标记TnC的单个半胱氨酸残基Cys-98，并在不存在和存在Ca ~（2+）和Mg ~（2+）以及其他肌钙蛋白亚基的情况下测定该探针对非离子猝灭剂丙烯酰胺（Eftink & Ghiron，1976）的可接近性。我们的研究表明标记的TnC，TnC*，
Paul C. Leavis,* Elizabeth Gowell, and Terence Tao abstract: Fluorescence lifetime and acrylamide quenching studies were carried out to characterize theinteractions between the subunits of troponinunder various conditions of metal ion binding. Troponin C was labeled at Cys-98 with 7V-(iodoacetyl)-7V-(5-sulfo-l-naphthyl) ethylenediamine. In the presence of Ca2+, the fluorescence decay of labeled troponin C (TnC*) was monoexponential, lifetime r= 15.5 ns and quenching rate constant kq= 2.97 X 10s M'1 s'1. In the absence of Ca2+, the decay was resolvable into a major com-ponent with= 11.9 ns and a minor component with= 20.5 ns, with corresponding values of kq= 4.80 X 10® and 0.66 X 10® M'1 s'1, respectively. Upon the binding of either troponin I (Tnl) or troponin T (TnT) in thepresence of Ca2+, r in-creased to~ 18 ns, and kq decreased to~ 0.8 X 10® M'1 s" 1.Regulation of vertebrate skeletal muscle contraction is triggered by Ca2+ binding to the troponin C (TnC) 1 moiety of the troponin complex which, in turn, is part of the thin filament. Metal-induced changes in the structure of TnC are thought to be transferred to the othertroponin subunits, Tnl and TnT, and ultimately to tropomyosin and actin to effect a change in the actin-myosin interaction. Although the details of this process are as yet unknown, the regulatory mechanism is likely to involve changes in protein-protein interaction in the filament that result in stronger binding between some of the filament components, eg, TnC and Tnl (Margossian & Cohen, 1973; Head & Perry, 1974; Potter & Gergely, 1974; Hitchcock, 1975; Ohnishi et al., 1975), and dissociation of others, eg, Tnl and actin (Margossian & Cohen, 1973; Potter & Gergely, 1974; Hitchcock, 1975). To further clarify the interactionsbetween the three troponin subunits, we have used the fluorescence quenching technique (Lehrer & Leavis, 1978; Eftink & Ghiron, 1981) to probe the microenvironment of the interacting sites. Changes in quenching parameters are interpretable in terms of conformational changes and changes in interprotein interactions involving the labeled protein. In this work, TnC was labeled at its single cysteine residue, Cys-98, with the fluorescent probe 1, 5-IAEDANS (Hudson & Weber, 1973), and theaccessi-bility of this probe to the nonionic quencher acrylamide (Eftink & Ghiron, 1976) was determined in the absence and presence of Ca2+ and Mg2+ and the other troponin subunits. Our studies indicate that labeled TnC, TnC*, exhibits small but distinct