Fluorescence studies of the interaction of calmodulin with myosin light chain kinase.

The interaction of calmodulin with myosin light chain kinase produces an approximately 30% increase in myosin light chain kinase tryptophan fluorescence. This represents the first report of calmodulin-induced structural changes in a protein which it activates. We fund that the calmodulin-myosin light chain kinase interaction is: 1) dependent on [Ca2+] (half-maximal binding at pCa 6.2) and essentially independent of [Mg2+], 2) occurs before saturation of all four reported Ca2+-specific sites on calmodulin. 3) saturates with 1 mol of calmodulin bound per mol of kinase with an apparent affinity of approximately 2.0 X 10(7) M-1, 4) is specific for calmodulin over troponin-C, 5) is directly related to the activation of myosin light chain kinase for phosphorylation of myosin light chain. Fluorescence stopped flow studies of these calmodulin-induced fluorescence changes in myosin light chain kinase indicate that Ca2+ binding to calmodulin occurs very rapidly and is not rate-limiting while the calmodulin-induced fluorescence increase in myosin light chain kinase occurs as a biphasic process with rates of approximately 65 s-1 and 6 s-1. The fluorescence increase produced by calmodulin binding to myosin light chain kinase is completely reversed by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at a rate of approximately 2 s-1.