Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces.
Surface-bound casein modulates the adsorption and activity of kinesin on SiO2 surfaces.
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DOI：
10.1016/j.bpj.2008.12.3960
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发表时间：
2009-04
影响因子：
3.4
通讯作者：
Tomomitsu Ozeki;V. Verma;M. Uppalapati;Yukiko Suzuki;Mikihiko Nakamura;J. Catchmark;W. Hancock
中科院分区：
文献类型：
--
作者：
Tomomitsu Ozeki;V. Verma;M. Uppalapati;Yukiko Suzuki;Mikihiko Nakamura;J. Catchmark;W. Hancock
常规的激动素通常被吸附到亲水性表面，如二氧化硅。用酪蛋白对表面进行预处理已成为获得最佳动蛋白活性的标准方案，但酪蛋白增强动蛋白表面吸附和功能的机制尚不清楚。我们使用石英晶体微天平和微管滑动分析来揭示酪蛋白在增强表面吸附的动蛋白活性方面所起的作用。在二氧化硅表面，酪蛋白以紧密结合的单分子层和可逆结合的第二层的形式吸附，其解离常数为500 nm，可以通过无酪素缓冲液洗涤来解吸。使用截断的动蛋白的实验表明，在存在可溶性酪蛋白的情况下，动蛋白的尾部与表面结合良好，而动蛋白的头部结合被阻止。去除可溶性酪蛋白会逆转这些结合图谱。令人惊讶的是，可逆结合的酪蛋白在动蛋白吸附过程中只起到了温和的作用，但当表面吸附的马达与微管相互作用时，它显著增强了动蛋白的活性。这些结果指出了一个模型，在该模型中，动态酪蛋白双层阻止了头部与表面的可逆关联，并增强了动蛋白尾部与表面的关联。理解这个模型体系中的蛋白质-表面相互作用应该为工程表面提供一个框架，以实现对其他马达蛋白和表面活性酶的功能性吸附。
Conventional kinesin is routinely adsorbed to hydrophilic surfaces such as SiO2. Pretreatment of surfaces with casein has become the standard protocol for achieving optimal kinesin activity, but the mechanism by which casein enhances kinesin surface adsorption and function is poorly understood. We used quartz crystal microbalance measurements and microtubule gliding assays to uncover the role that casein plays in enhancing the activity of surface-adsorbed kinesin. On SiO2surfaces, casein adsorbs as both a tightly bound monolayer and a reversibly bound second layer that has a dissociation constant of 500 nM and can be desorbed by washing with casein-free buffer. Experiments using truncated kinesins demonstrate that in the presence of soluble casein, kinesin tails bind well to the surface, whereas kinesin head binding is blocked. Removing soluble casein reverses these binding profiles. Surprisingly, reversibly bound casein plays only a moderate role during kinesin adsorption, but it significantly enhances kinesin activity when surface-adsorbed motors are interacting with microtubules. These results point to a model in which a dynamic casein bilayer prevents reversible association of the heads with the surface and enhances association of the kinesin tail with the surface. Understanding protein-surface interactions in this model system should provide a framework for engineering surfaces for functional adsorption of other motor proteins and surface-active enzymes.