Distances between tropomyosin sites across the muscle thin filament using luminescence resonance energy transfer: evidence for tropomyosin flexibility.
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.
Chen
Yaodong
Y
Muscle and Motility Group, 64 Grove Street, Boston Biomedical Research Institute, Watertown, Massachusetts 02472, USA. y.chen@cellbio.duke.edu
Lehrer
Sherwin S
SS
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
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