Regulatory properties of recombinant tropomyosins containing 5-hydroxytryptophan: Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site.
We have introduced tryptophan codons at different positions of the chicken alpha-tropomyosin cDNA (Monteiro, P. B., Lataro, R. C., Ferro, J. A., and Reinach, F. C. (1994) J. Biol. Chem. 269, 10461-10466) and employed a trp auxotrophic Escherichia coli strain to express the proteins in media containing either normal tryptophan, 5-hydroxytrptophan, or 7-azatryptophan. The fluorescence of these latter two tryptophan analogues is excitable at 312-315 nm at which the natural fluorescence of other thin filament proteins (actin, troponin) is not excited. The recombinant tropomyosins have tryptophans or analogues located at amino acid positions 90, 101, 111, 122, or 185 of the protein, all on the external surface of the tropomyosin coiled-coil (positions "c" or "f" of the hydrophobic heptad repeat). The first four mutations are located within the third actin-binding zone of tropomyosin, a region not expected to interact directly with troponin or with neighboring tropomyosin molecules in muscle thin filaments, while position 185 is located in a region that has been implicated in interactions with the globular domain of troponin. The fluorescence intensity of the mutant containing 5-hydroxytryptophan at position 122 (5OH122W) is sensitive to actin binding and sensitive to Ca2+-binding to thin filaments reconstituted with troponin. Assuming that the globular domain of troponin binds to a site between residues 150 and 190 of tropomyosin, the distance between the troponin-binding site and the fluorescent probes at position 122 can be estimated to be 4.2-10.2 nm. While X-ray diffraction and electron micrograph reconstitution studies have provided evidence of Ca2+-induced changes in tropomyosin's interactions in the thin filament, their resolution was not sufficient to distinguish between changes involving the whole tropomyosin molecule or only that region directly interacting with troponin. Here we provide a clear demonstration that Ca2+-binding to troponin results in a conformational change in a region of tropomyosin outside the troponin binding site which is probably associated with a changed interaction with actin.
Farah
C S
CS
Departamento de Bioquímica, Instituto de Química, Howard Hughes Medical Institute, Universidade de São Paulo, Brazil. chuck@iris1.iq.usp.br
Reinach
F C
FC
D006916:5-Hydroxytryptophan / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000037:antagonists & inhibitors; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014336:Troponin / Q000737:chemistry / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives / Q000235:genetics
D000199:Actins; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; C006657:7-azatryptophan; D014364:Tryptophan; D000205:Actomyosin; D006916:5-Hydroxytryptophan; D009218:Myosins; D002118:Calcium