Chevreux Guillaume, Potier Noelle, Van Dorsselaer Alain, Bahloul Amel, Houdusse Anne, Wells Amber, Sweeney H Lee
Laboratoire de Spectrométrie de Masse Bio-Organique, Strasbourg, France.
J Am Soc Mass Spectrom. 2005 Aug;16(8):1367-76. doi: 10.1016/j.jasms.2005.03.023.
Among the myosin superfamily, myosin VI differs from all others by a reverse directionality and a particular motility. Little structural information is available for myosin VI. It is known that it binds one calmodulin (CaM) by means of a single "IQ motif" and that myosin VI contains a specific insert located at the junction between the motor domain (MD) and the lever arm, likely to play a critical role for the unusual motility previously observed. Electrospray ionization mass spectrometry (MS) was used to determine the CaM and Ca2+ stoichiometries in several myosin VI constructs. In particular, the experimental conditions required for the observation of multiprotein/Ca2+ noncovalent assemblies are detailed for two truncated MD constructs (less than 20 kDa) and for three full MD constructs (more than 90 KDa). The specificity of the detected stoichiometries is discussed for each construct and the resolving power of Time of Flight mass spectrometry is stressed, in particular for the detection of metal ions binding to high molecular weight complexes. MS reveals a new CaM binding site for myosin VI and highlights a different behavior for the five myosin VI constructs versus Ca2+ binding. In addition to these stoichiometry based experiments, gas-phase dissociation analyses on intact complexes are described. They reveal that Ca2+ transfer between protein partners occurs during the dissociation process for one construct with a full MD. Charge-transfer and dissociation behavior has allowed to draw structural assumptions for the interaction of the MD with the CaM N-terminal lobe.
在肌球蛋白超家族中，肌球蛋白VI与其他所有成员的不同之处在于其反向运动性和独特的运动方式。目前关于肌球蛋白VI的结构信息较少。已知它通过单个“IQ基序”结合一个钙调蛋白（CaM），并且肌球蛋白VI在马达结构域（MD）和杠杆臂之间的连接处含有一个特定插入序列，这可能对先前观察到的异常运动性起着关键作用。采用电喷雾电离质谱（MS）来确定几种肌球蛋白VI构建体中的CaM和Ca2+化学计量。特别是，详细介绍了观察两种截短的MD构建体（小于20 kDa）和三种完整MD构建体（大于90 kDa）的多蛋白/Ca2+非共价组装所需的实验条件。针对每种构建体讨论了检测到的化学计量的特异性，并强调了飞行时间质谱的分辨能力，特别是对于检测与高分子量复合物结合的金属离子。MS揭示了肌球蛋白VI的一个新的CaM结合位点，并突出了五种肌球蛋白VI构建体在Ca2+结合方面的不同行为。除了这些基于化学计量的实验外，还描述了对完整复合物的气相解离分析。结果表明，对于一种具有完整MD的构建体，在解离过程中蛋白伴侣之间会发生Ca2+转移。电荷转移和解离行为有助于对MD与CaM N端叶相互作用的结构进行假设。
