Ca(2+)-dependent, myosin subfragment 1-induced proximity changes between actin and the inhibitory region of troponin I.
In order to help understand the spatial rearrangements of thin filament proteins during the regulation of muscle contraction, we used fluorescence resonance energy transfer (FRET) to measure Ca(2+)-dependent, myosin-induced changes in distances and fluorescence energy transfer efficiencies between actin and the inhibitory region of troponin I (TnI). We labeled the single Cys-117 of a mutant TnI with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IAEDANS) and Cys-374 of actin with 4-dimethylaminophenylazophenyl-4'-maleimide (DABmal). These fluorescent probes were used as donor and acceptor, respectively, for the FRET measurements. We reconstituted a troponin-tropomyosin (Tn-Tm) complex which contained the AEDANS-labeled mutant TnI, together with natural troponin T (TnT), troponin C (TnC) and tropomyosin (Tm) from rabbit fast skeletal muscle. Fluorescence titration of the AEDANS-labeled Tn-Tm complex with DABmal-labeled actin, in the presence and absence of Ca(2+), resulted in proportional, linear increases in energy transfer efficiency up to a 7:1 molar excess of actin over Tn-Tm. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased from 37.9 A to 44.1 A when Ca(2+) bound to the regulatory sites of TnC. Titration of reconstituted thin filaments, containing AEDANS-labeled Tn-Tm and DABmal-labeled actin, with myosin subfragment 1 (S1) decreased the energy transfer efficiency, in both the presence and absence of Ca(2+). The maximum decrease occurred at well below stoichiometric levels of S1 binding to actin, showing a cooperative effect of S1 on the state of the thin filaments. S1:actin molar ratios of approximately 0.1 in the presence of Ca(2+), and approximately 0.3 in the absence of Ca(2+), were sufficient to cause a 50% reduction in normalized transfer efficiency. The distance between AEDANS on TnI Cys-117 and DABmal on actin Cys-374 increased by approximately 7 A in the presence of Ca(2+) and by approximately 2 A in the absence of Ca(2+) when S1 bound to actin. Our results suggest that TnI's interaction with actin inhibits actomyosin ATPase activity by modulating the equilibria among active and inactive states of the thin filament. Structural rearrangements caused by myosin S1 binding to the thin filament, as detected by FRET measurements, are consistent with the cooperative behavior of the thin filament proteins.
Kobayashi
T
T
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
Kobayashi
M
M
Collins
J H
JH
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D004735:Energy Transfer; D005456:Fluorescent Dyes; D018482:Muscle, Skeletal / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry; D009282:Naphthalenesulfonates; D011817:Rabbits; D019210:Troponin I / Q000737:chemistry*
D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009282:Naphthalenesulfonates; D019210:Troponin I; C048876:1,5-AEDANS; D009218:Myosins; D002118:Calcium