Fluorescence Lifetime Imaging Reveals that the Environment of the ATP Binding Site of Myosin in Muscle Senses Force
Fluorescence Lifetime Imaging Reveals that the Environment of the ATP Binding Site of Myosin in Muscle Senses Force
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荧光寿命成像揭示肌肉感觉力中肌球蛋白 ATP 结合位点的环境
DOI：
10.1016/j.bpj.2010.07.052
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发表时间：
2010
影响因子：
3.4
通讯作者：
Ibanez-Garcia D
中科院分区：
文献类型：
--
作者：
Ibanez-Garcia D
荧光寿命成像显微镜被用来证明施加在肌肉纤维上的不同载荷改变了肌球蛋白核苷酸结合口袋的微环境。用荧光三磷酸腺苷类似物3‘-二乙酸丙二胺-三磷酸腺苷(3′-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP)，标记僵直的通透性骨骼肌纤维，并将其水解成二磷酸。小幅度拉伸和释放的循环(肌节长度的1%)与荧光寿命成像和FORCE测量同步，以关联力对结合到肌动肌球蛋白复合体上的ATP类似物寿命的影响。荧光衰变分析可分辨出两个寿命，分别对应于游离核苷酸DEAc-PDA-ATP(τ1=0.47±0.0 3 ns；平均值±SD)和与肌球蛋白复合体结合的核苷酸(低应变时τ2=2.2 1±0.0 6 ns)。τ-1不随力的变化而变化，而τ-2与施加在肌肉上的力呈线性关系，为0.43±0.05ps/kpa。因此，肌球蛋白核苷酸结合口袋的分子环境直接受到施加在纤维片段末端的长度变化的影响。这些变化可能有助于解释力量如何调节肌动球蛋白ATPase循环，从而调节收缩的生理学和能量学。
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). Whereasτ1did not change with force,τ2showed 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.