title: Calcium-induced quenching of intrinsic fluorescence in brain myosin V is linked to dissociation of calmodulin light chains.
abstract: Myosin V isolated from chick brain (BM V) is a multimeric protein of about 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 heavy chain is an extended neck region with six consensus IQ sites for the binding of calmodulin (CaM) and myosin light chains. The actin-activated MgATPase has been shown to require >/=1 microM Ca2+ for full activity, and evidence points to a myosin-linked regulatory system where the CaM light chains participate as modulators for the Ca2+ signal. Still, the precise mechanism of Ca2+ regulation remains unknown. In the present study we have used the intrinsic tryptophan fluorescence of native BM V to monitor conformational changes of BM V induced by Ca2+, and we relate these changes to CaM dissociation from the BM V molecule. The fluorescence intensity decreases approximately 17% upon addition of sub-micromolar concentrations of Ca2+ (K0.5 = 0.038 microM). This decrease in fluorescence, which is dominated by a conformational 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 filtration of native BM V using HPLC shows that CaM is partially dissociated from the heavy chain in EGTA and dissociates further upon addition of sub-micromolar concentrations of Ca2+. These observations suggest that the affinity of CaM for at least one of the IQ sites on the BM V heavy chain decreases with Ca2+ and that the Ca2+ concentration required for this effect is lower than that needed to activate acto-BM V. Using a cosedimentation assay in the presence of actin, we also observe partial 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 when Ca2+ exceeds 5-10 microM. Thus, the binding of CaM to BM V is affected by both actin and Ca2+.
authors.affiliation: Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941-590 RJ, Brazil.
authors.forename: L C
authors.initials: LC
authors.lastname: Cameron
authors.forename: R N
authors.initials: RN
authors.lastname: Carvalho
authors.forename: J R
authors.initials: JR
authors.lastname: Araujo
authors.forename: A C
authors.initials: AC
authors.lastname: Santos
authors.forename: S B
authors.initials: SB
authors.lastname: Tauhata
authors.forename: R E
authors.initials: RE
authors.lastname: Larson
authors.forename: M M
authors.initials: MM
authors.lastname: Sorenson
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D005453:Fluorescence; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008970:Molecular Weight; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D013050:Spectrometry, Fluorescence
chemical_list: D000199:Actins; D002147:Calmodulin; D046911:Macromolecular Substances; D009218:Myosins; D002118:Calcium