Sun Mingxuan, Oakes Judy L, Ananthanarayanan Shobana K, Hawley Katherine H, Tsien Roger Y, Adams Stephen R, Yengo Christopher M
Department of Biology, University of North Carolina, Charlotte, NC 28223, USA.
J Biol Chem. 2006 Mar 3;281(9):5711-7. doi: 10.1074/jbc.M508103200. Epub 2005 Dec 23.
The upper 50-kDa region of myosin may be critical for coupling between the nucleotide- and actin-binding regions. We introduced a tetracysteine motif in the upper 50-kDa domain (residues 292-297) of myosin V containing a single IQ domain (MV 1IQ), allowing us to label this site with the fluorescein biarscenical hairpin-binding dye (FlAsH) (MV 1IQ FlAsH). The enzymatic properties of MV 1IQ FlAsH were similar to those of unlabeled MV 1IQ except for a 3-fold reduced ADP-release rate. MV 1IQ FlAsH was also capable of moving actin filaments in the in vitro motility assay. To examine rotation of the upper 50-kDa region, we determined the difference in the degree of energy transfer from N-methylanthraniloyl (mant)-labeled nucleotides to FlAsH in both steady-state and transient kinetic experiments. The energy transfer efficiency was higher with mant-ATP (0.65 +/- 0.02) compared with mant-ADP (0.55 +/- 0.02) in the absence of actin. Stopped-flow measurements suggested that the energy transfer efficiency decreased with phosphate release (0.04 s(-1)) in the absence of actin. In contrast, upon mixing MV 1IQ FlAsH in the ADP.P(i) state with actin, a decrease in the energy transfer signal was observed at a rate of 13 s(-1), similar to the ADP release rate. Our results demonstrate there was no change in the energy transfer signal upon actin-activated phosphate release and suggest that actin binding alters the dynamics of the upper 50-kDa region, which may be critical for the ability of myosin to bind tightly to both ADP and actin.
肌球蛋白50 kDa以上的区域可能对核苷酸结合区域和肌动蛋白结合区域之间的偶联至关重要。我们在含有单个IQ结构域的肌球蛋白V的50 kDa以上结构域（第292 - 297位氨基酸残基）中引入了一个四半胱氨酸基序，使我们能够用荧光素双砷酸发夹结合染料（FlAsH）标记该位点（MV 1IQ FlAsH）。MV 1IQ FlAsH的酶学性质与未标记的MV 1IQ相似，只是ADP释放速率降低了3倍。在体外运动分析中，MV 1IQ FlAsH也能够使肌动蛋白丝移动。为了检测50 kDa以上区域的旋转，我们在稳态和瞬态动力学实验中测定了从N - 甲基邻氨基苯甲酰（mant）标记的核苷酸到FlAsH的能量转移程度的差异。在没有肌动蛋白的情况下，与mant - ADP（0.55±0.02）相比，mant - ATP的能量转移效率更高（0.65±0.02）。停流测量表明，在没有肌动蛋白的情况下，随着磷酸盐释放（0.04 s⁻¹），能量转移效率降低。相反，当处于ADP·P(i)状态的MV 1IQ FlAsH与肌动蛋白混合时，观察到能量转移信号以13 s⁻¹的速率下降，这与ADP释放速率相似。我们的结果表明，肌动蛋白激活的磷酸盐释放后能量转移信号没有变化，并表明肌动蛋白结合改变了50 kDa以上区域的动力学，这可能对肌球蛋白紧密结合ADP和肌动蛋白的能力至关重要。
