Burghardt Thomas P, Ajtai Katalin, Chan Daniel K, Halstead Miriam F, Li Jinhui, Zheng Ye
Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
Biophys J. 2007 Sep 15;93(6):2226-39. doi: 10.1529/biophysj.107.107433. Epub 2007 May 18.
Myosin is the molecular motor in muscle-binding actin and executing a power stroke by rotating its lever arm through an angle of approximately 70 degrees to translate actin against resistive force. A green fluorescent protein (GFP)-tagged human cardiac myosin regulatory light chain (HCRLC) was constructed to study in situ lever arm orientation one molecule at a time by polarized fluorescence emitted from the GFP probe. The recombinant protein physically and functionally replaced the native RLC on myosin lever arms in the thick filaments of permeabilized skeletal muscle fibers. Detecting single molecules in fibers where myosin concentration reaches 300 microM is accomplished using total internal reflection fluorescence microscopy. With total internal reflection fluorescence, evanescent field excitation, supercritical angle fluorescence detection, and CCD detector pixel size limits detection volume to just a few attoliters. Data analysis manages both the perturbing effect of the TIR interface on probe emission and the effect of high numerical aperture collection of light. The natural myosin concentration gradient in a muscle fiber allows observation of fluorescence polarization from C-term GFP-tagged HCRLC exchanged myosin from regions in the thick filament containing low and high myosin concentrations. In rigor, cross-bridges at low concentration at the end of the thick filament maintain GFP dipole moments at two distinct polar angles relative to the fiber symmetry axis. The lower angle, where the dipole is nearly parallel to fiber axis, is more highly populated than the alternative, larger angle. Cross-bridges at higher concentration in the center of the thick filament are oriented in a homogeneous band at approximately 45 degrees to the fiber axis. The data suggests molecular crowding impacts myosin conformation, implying mutual interactions between cross-bridges alter how the muscle generates force. The GFP-tagged RLC is a novel probe to assess single-lever-arm orientation characteristics in situ.
肌球蛋白是肌肉中的分子马达，它与肌动蛋白结合，并通过将其杠杆臂旋转约70度来执行动力冲程，从而克服阻力使肌动蛋白移动。构建了一种绿色荧光蛋白（GFP）标记的人心脏肌球蛋白调节轻链（HCRLC），以便通过GFP探针发出的偏振荧光一次研究一个分子的原位杠杆臂方向。重组蛋白在功能和物理上替代了通透化骨骼肌纤维粗肌丝中肌球蛋白杠杆臂上的天然RLC。使用全内反射荧光显微镜可以检测肌球蛋白浓度达到300 microM的纤维中的单个分子。通过全内反射荧光、倏逝场激发、超临界角荧光检测以及CCD探测器像素大小，将检测体积限制在仅几飞升。数据分析处理了TIR界面对探针发射的干扰效应以及高数值孔径光收集的效应。肌肉纤维中天然的肌球蛋白浓度梯度使得能够观察到从粗肌丝中含有低和高肌球蛋白浓度区域交换而来的C端GFP标记的HCRLC的肌球蛋白的荧光偏振。在僵直状态下，粗肌丝末端低浓度的横桥相对于纤维对称轴在两个不同的极角保持GFP偶极矩。偶极几乎与纤维轴平行的较小角度比另一个较大角度的数量更多。粗肌丝中心较高浓度的横桥以与纤维轴成约45度的均匀带排列。数据表明分子拥挤会影响肌球蛋白构象，这意味着横桥之间的相互作用会改变肌肉产生力的方式。GFP标记的RLC是一种评估原位单杠杆臂方向特征的新型探针。
