Cross-linking and fluorescence study of the COOH- and NH2-terminal domains of intact caldesmon bound to actin.
The NH2- and COOH-terminal domains of muscle caldesmon are separated by a long alpha-helical stretch. Cys-580, in the COOH-terminal domain, can be rapidly and efficiently disulfide-cross-linked to Cys-374 of actin by incubation with actin modified with 5,5'-dithiobis(2-nitrobenzoic acid) (Graceffa, P., and Jancso, A. (1991) J. Biol. Chem. 266, 20305-20310). Upon further incubation (+/- tropomyosin), a second cross-link was slowly formed between Cys-153 in the NH2-terminal domain and Cys-374 of another actin monomer. The yield of the second cross-link was relatively insensitive to increasing ionic strength, whereas the caldesmon-actin binding strength decreased considerably, suggesting that the NH2-terminal domain is largely dissociated from actin and becomes slowly cross-linked to it during collisions with the actin filament. In support of these conclusions, the yield of photocross-linking actin to caldesmon specifically labeled with benzophenonemaleimide at Cys-580 was high, but close to zero for caldesmon labeled at Cys-153, and the fluorescence intensity and polarization of tetramethylrhodamine iodoacetamide attached to Cys-580 showed large changes, while there were no changes for the probe at Cys-153 upon binding caldesmon to actin (+/- tropomyosin). These findings are consistent with the knowledge that COOH-terminal fragments of caldesmon bind to actin, whereas NH2-terminal fragments do not. Since the NH2-terminal domain of caldesmon binds to myosin, a dissociated NH2-terminal domain may account for caldesmon's ability to link myosin and actin filaments.
Graceffa
P
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Muscle Research Group, Boston Biomedical Research Institute, Massachusetts 02114, USA.
D000199:Actins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002148:Calmodulin-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D003432:Cross-Linking Reagents; D003545:Cysteine; D004228:Dithionitrobenzoic Acid; D005895:Gizzard, Avian; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010618:Phenanthrolines / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000199:Actins; D002148:Calmodulin-Binding Proteins; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010618:Phenanthrolines; D004228:Dithionitrobenzoic Acid; D003545:Cysteine; C025205:1,10-phenanthroline