Cai Dawen, Hoppe Adam D, Swanson Joel A, Verhey Kristen J
Biophysics Research Division, University of Michigan, Ann Arbor, MI 48109, USA.
J Cell Biol. 2007 Jan 1;176(1):51-63. doi: 10.1083/jcb.200605097.
Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)-labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.
驱动蛋白运动蛋白沿着微管轨道驱动细胞货物的运输。运动蛋白活性在细胞中是如何被控制的尚未解决，但它可能与蛋白质构象和货物结合的变化相关。通过将荧光共振能量转移（FRET）化学计量学定量方法应用于活细胞中荧光蛋白（FP）标记的驱动蛋白重链（KHC）和驱动蛋白轻链（KLC）亚基，我们研究了驱动蛋白-1在活性和非活性状态下的整体结构组织和构象。非活性驱动蛋白-1分子折叠并自抑制，使得KHC尾部阻断KHC运动蛋白与微管的初始相互作用。此外，在非活性状态下，KHC运动结构域被KLC亚基推开。因此，FRET化学计量学揭示了活细胞中蛋白质复合物的构象变化。对于驱动蛋白-1，激活需要一个全局构象变化，将KHC运动结构域和尾部结构域分开，以及一个局部构象变化，使KHC运动结构域靠得更近。
