title: Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility.
abstract: To obtain information about the interaction of tropomyosin (Tm) with actin associated with the regulatory states of the muscle thin filament, we used luminescence resonance energy transfer (LRET) between Tb(3+) as a donor and rhodamine as an acceptor. A novel Tb(3+) chelator, S-(2-nitro-5-thiobenzoate)cysteaminyl-DTPA-Cs124, was synthesized, which specifically labels Cys groups in proteins. With the Tb chelate as the donor and tetramethylrhodamine-5-maleimide as the acceptor, both bound to specific Cys groups of Tm, we obtained 67 A as the distance between Tm's across the actin filament, a much shorter value than that obtained from structural studies (72-86 A). The difference appears to be due to submillisecond motion associated with Tm flexibility, which brings the probes closer during the millisecond lifetime of the donor. Ca(2+) did not change the energy transfer with the reconstituted thin filament, but myosin subfragment 1 decreased the transfer, consistent with either a 5-6 A increase in distance or, more likely, a decrease in flexibility.
authors.affiliation: Muscle and Motility Group, 64 Grove Street, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA. y.chen@cellbio.duke.edu
authors.forename: Yaodong
authors.initials: Y
authors.lastname: Chen
authors.forename: Sherwin S
authors.initials: SS
authors.lastname: Lehrer
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000818:Animals; D002645:Chickens; D004492:Edetic Acid / Q000737:chemistry; D005470:Fluorometry; D007700:Kinetics; D018499:Linear Energy Transfer / Q000235:genetics; D008163:Luminescent Measurements; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry* / Q000378:metabolism; D015879:Myosin Subfragments / Q000378:metabolism; D009579:Nitrobenzoates / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D004369:Pentetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014336:Troponin / Q000378:metabolism
chemical_list: D000199:Actins; D015879:Myosin Subfragments; C121732:NTB-cysteaminyl-EDTA; D009579:Nitrobenzoates; D009942:Organometallic Compounds; D014335:Tropomyosin; D014336:Troponin; C113011:europium DTPA-cs124; D004369:Pentetic Acid; D004492:Edetic Acid