CALCIUM-INDUCED QUENCHING OF INTRINSIC FLUORESCENCE IN BRAIN MYOSIN-VIS LINKED TO DISSOCIATION OF CALMODULIN LIGHT-CHAINS

Myosin V isolated from chick brain (BM V) is a multimeric protein of a bout 640 kDa consisting of two intertwined heavy chains of 212 kDa and multiple light chains of 10 to 20 kDa. A distinctive feature of the h eavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin Light chains. The actin-act ivated MgATPase has been shown to require greater than or equal to 1 m u M Ca2+ for full activity, and evidence points to a myosin-linked reg ulatory system where the CaM light chains participate as modulators fo r the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation rem ains unknown. In the present study we have used the intrinsic tryptoph an fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation fr om the BM V molecule. The fluorescence intensity decreases similar to 17% upon addition of sub-micromolar concentrations of Ca2+ (K-0.5 = 0. 038 mu M). This decrease in fluorescence, which is dominated by a conf ormational change in the heavy chain, can be reversed by addition of 1 ,2-di(2-aminoethoxy)ethane-N,N,N',N'- tetraacetic acid (EGTA) followed by an excess of CaM, but not by addition of EGTA alone. Gel filtratio n of native BM V using HPLC shows that CaM is partially dissociated fr om the heavy chain in EGTA and dissociates further upon addition of su b-micromolar concentrations of Ca2+. These observations suggest that t he affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with CA(2+) and that the Ca2+ concentration required f or this effect is lower than that needed to activate acto-BM V, Using a cosedimentation assay in the presence of actin, we also observe part ial dissociation of CaM: when Ca2+ is absent, but now the addition of Ca2+ has a biphasic effect: sub-micromolar Ca2+ concentrations lead to reassociation of CaM with the heavy chain, followed by dissociation w hen Ca2+ exceeds 5-10 mu M. Thus, the binding of CaM to BM V is affect ed by both actin and Ca2+. (C) 1998 Academic Press.