Ca2+-induced rolling of tropomyosin in muscle thin filaments: the alpha- and beta-band hypothesis revisited.
Tropomyosin is a filamentous coiled-coil protein directly involved in the regulation of the actomyosin interaction responsible for muscle contraction: it transmits the local calcium-induced conformational change in troponin to the helical array of myosin-binding sites on the surface of the actin filament. McLachlan and Stewart (McLachlan, A. D., and Stewart, M. (1976) J. Mol. Biol. 103, 271-298) proposed that the tropomyosin coiled-coil structure can be divided into 14 alternating 19- to 20-residue "alpha- and beta-bands," which could act as alternate 7-fold sets of sites for specific binding to actin in the different conformational states of the regulated thin filament. Here we present the first direct experimental evidence in support of the alpha- and beta-band hypothesis: we analyze the acrylamide quenching of the fluorescence of mutant tropomyosins containing 5-hydroxytryptophan residues at different positions along the coiled-coil structure under a variety of conditions (alone, complexed with actin, and complexed with actin and troponin with or without Ca(2+)). We show that fluorescent probes placed in the alpha-bands become less solvent-exposed in the absence of calcium, whereas those in the beta-bands become less solvent-exposed in the presence of calcium. A model in which the tropomyosin coiled-coil rolls across the actin surface in response to Ca(2+)-binding to troponin most easily explains these observations.
Holthauzen
Luis M F
LM
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, CEP 05508-900, São Paulo, SP, Brazil. chsfarah@iq.usp.br
Corrêa
Fernando
F
Farah
Chuck S
CS
D006916:5-Hydroxytryptophan / Q000737:chemistry; D020106:Acrylamide / Q000494:pharmacology; D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000737:chemistry; D000205:Actomyosin / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014335:Tropomyosin / Q000737:chemistry*
D000199:Actins; D014335:Tropomyosin; D020106:Acrylamide; D000205:Actomyosin; D006916:5-Hydroxytryptophan; D000251:Adenosine Triphosphatases; D009218:Myosins; D002118:Calcium