Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization
Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization
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DOI：
10.1021/bi962392l
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发表时间：
1997-02-18
期刊：
影响因子：
2.9
通讯作者：
Shimizu, T
中科院分区：
文献类型：
--
作者：
Morii, H;Takenawa, T;Shimizu, T
驱动蛋白重链由称为马达结构域的N-末端球状结构域、杆状中间区域和C-末端结构域组成。在这项研究中，人驱动蛋白颈部区域，该区域邻近的运动域，促进二聚化，已被调查。首先，我们预测卷曲螺旋区域，包括颈部区域，我们新设计的统计方法。序列（335-372）由独特的七肽两亲性主导。通过圆二色性（CD）光谱比较细菌表达的人驱动蛋白重链片段K349（1-349）（单体马达结构域）和K379（1-379）（二聚体），表明K379具有更多的α-螺旋含量。化学合成的肽（332-349）、（350-379）和（332-369）给出了富含α-螺旋模式的CD光谱，但光谱随肽浓度而变化。在222 nm处的摩尔椭圆率分析表明，这些肽处于单体-二聚体平衡，并且解离等温线分别为9.6 mM、60 μ M和62 nM建立了上述肽的解离常数。沉降平衡测量证实肽（332-369）以二聚体形式存在。这些结果有力地表明，从332到369的颈部区域的序列形成一个ct螺旋卷曲螺旋。K349和K379的差异肽（350-379）没有显示出使K379二聚的足够能力。区域（350-379）可能仅与（332-349）区域一起形成稳定的α-螺旋卷曲螺旋。用荧光供体和受体标记的[Cys（363）]-（332-369）的荧光能量转移研究表明，该肽形成平行卷曲螺旋。这种卷曲螺旋对尿素和热变性是热稳定的，并且在中性pH下，卷曲螺旋的肽交换是不可检测的，或非常缓慢。在22 μ M的肽浓度下，解离自由能估计为57.7 kJ mol（-1）。这些结果表明，驱动蛋白的颈部区域形成一个稳定的卷曲螺旋，这可能是重要的二聚体驱动蛋白的运动。
The kinesin heavy chain consists of an N-terminal globular domain, referred to as the motor domain, a rod-like middle region, and a C-terminal domain. In this study, the human kinesin neck region, the region adjacent to the motor domain which promotes dimerization, has been investigated. First, we predicted coiled-coil regions including the neck region by our newly devised statistical method. The sequence (335-372) was predominated by a unique heptad amphipathy. A comparison of the bacterially expressed human kinesin heavy chain fragments, K349 (1-349), a monomeric motor domain, and K379 (1-379), a dimer, by circular dichroism (CD) spectroscopy showed that K379 had more alpha-helical content. Chemically synthesized peptides, (332-349), (350-379), and (332-369), gave CD spectra with an alpha-helix-rich pattern, but the spectra varied depending on the peptide concentration. Analysis of the molar ellipticity at 222 nm indicated that those peptides were in monomer-dimer equilibria, and the dissociation isotherms established dissociation constants of 9.6 mM, 60 mu M, and 62 nM for the above peptides, respectively. Sedimentation equilibrium measurements verified that the peptide (332-369) existed as a dimeric form. These results strongly suggest that the sequence from 332 to 369 of the neck region forms an ct-helical coiled coil. The differential peptide of K349 and K379, (350-379), did not show sufficient ability to make K379 dimeric. It is likely that the region (350-379) forms a stable alpha-helical coiled coil only together with the (332-349) region. Fluorescence energy transfer studies of [Cys(363)]-(332-369) labeled with a fluorescence donor and an acceptor revealed that the peptide formed a parallel coiled coil. This coiled coil was thermodynamically stable against urea and thermal denaturation, and peptide exchange of the coiled coil was undetectable, or extremely slow, at neutral pH. The dissociation free energy was estimated to be 57.7 kJ mol(-1) at a peptide concentration of 22 mu M. These results indicate that the neck region of kinesin forms a stable coiled coil which may be important for the motility of dimeric kinesin.