Fluorescence lifetime imaging reveals that the environment of the ATP binding site of myosin in muscle senses force |
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Authors: | Ibanez-Garcia Delisa Requejo-Isidro Jose Webb Martin R West Timothy G French Paul Ferenczi Michael A |
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Institution: | † Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom ‡ Photonics Group, Physics Department, Imperial College London, London, United Kingdom § Unidad de Biofisica, Consejo Superior de Investigaciones Científicas, University of the Basque Country, Leioa, Spain ¶ Medical Research Council, National Institute for Medical Research, London, United Kingdom |
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Abstract: | Fluorescence lifetime imaging microscopy is used to demonstrate that different loads applied to a muscle fiber change the microenvironment of the nucleotide binding pocket of myosin. Permeabilized skeletal muscle fibers in rigor were labeled with a fluorescent ATP analog, 3′-DEAC-propylenediamine (pda)-ATP (3′-O-{N-3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP), which was hydrolyzed to the diphosphate. Cycles of small-amplitude stretches and releases (<1% of muscle segment length) were synchronized with fluorescence lifetime imaging and force measurements to correlate the effect of force on the lifetime of the ATP analog bound to the actomyosin complex. Analysis of the fluorescence decay resolved two lifetimes, corresponding to the free nucleotide DEAC-pda-ATP (τ1 = 0.47 ± 0.03 ns; mean ± SD) and nucleotide bound to the actomyosin complex (τ2 = 2.21 ± 0.06 ns at low strain). Whereas τ1 did not change with force, τ2 showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kPa. Hence, the molecular environment of the nucleotide binding pocket of myosin is directly affected by a change of length applied at the ends of the fiber segments. These changes may help explain how force modulates the actomyosin ATPase cycle and thus the physiology and energetics of contraction. |
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