title: Characterisation of a mutant of barnacle troponin C lacking Ca2+-binding sites at positions II and IV.
abstract: This study investigates a mutant barnacle troponin C (TnC) protein (BTnC2-4-) in which the Ca2+-binding sites (sites II and IV) have been rendered non-functional. Eliminating Ca2+ binding at both Ca2+-binding sites of barnacle TnC did not prevent the incorporation of BTnC2-4- into TnC-depleted myofibrillar bundles, although, as expected, the mutant was not able to effect muscle regulation. We conclude that the Mg2+ involved in stabilising the interaction between TnC and TnI in the barnacle must bind at a separate location to the Ca2+-binding sites. Competition experiments between BTnC2-4- and wild-type barnacle TnC (BTnCWT) or the native isoform BTnC2 indicate that BTnC2-4- has an approximately fourfold higher affinity for barnacle TnI than BTnCWT but a lower affinity for TnI compared to BTnC2. These results indicate that disabling sites II and IV changes the affinity of BTnC2-4- for TnC-denuded barnacle myofibrils, altering the stability of the bond formed between TnC and the thin filament.
authors.affiliation: University Laboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK.
authors.forename: L D
authors.initials: LD
authors.lastname: Allhouse
authors.forename: G
authors.initials: G
authors.lastname: Guzman
authors.forename: T
authors.initials: T
authors.lastname: Miller
authors.forename: Q
authors.initials: Q
authors.lastname: Li
authors.forename: J D
authors.initials: JD
authors.lastname: Potter
authors.forename: C C
authors.initials: CC
authors.lastname: Ashley
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007537:Isometric Contraction / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009210:Myofibrils / Q000378:metabolism; D009412:Nerve Fibers / Q000378:metabolism; D011485:Protein Binding; D001468:Thoracica / Q000235:genetics* / Q000378:metabolism*; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D002135:Calcium-Binding Proteins; D019209:Troponin C; D002118:Calcium