School of Natural Sciences, The Linnaeus University SE-391 82 Kalmar, Sweden.
Langmuir. 2010 Jun 15;26(12):9927-36. doi: 10.1021/la100395a.
In the in vitro motility assay, actin filaments are propelled by surface-adsorbed myosin motors, or rather, myosin motor fragments such as heavy meromyosin (HMM). Recently, efforts have been made to develop actomyosin powered nanodevices on the basis of this assay but such developments are hampered by limited understanding of the HMM adsorption geometry. Therefore, we here investigate the HMM adsorption geometries on trimethylchlorosilane- [TMCS-] derivatized hydrophobic surfaces and on hydrophilic negatively charged surfaces (SiO(2)). The TMCS surface is of great relevance in fundamental studies of actomyosin and both surface substrates are important for the development of motor powered nanodevices. Whereas both the TMCS and SiO(2) surfaces were nearly saturated with HMM (incubation at 120 microg mL(-1)) there was little actin binding on SiO(2) in the absence of ATP and no filament sliding in the presence of ATP. This contrasts with excellent actin-binding and motility on TMCS. Quartz crystal microbalance with dissipation (QCM-D) studies demonstrate a HMM layer with substantial protein mass up to 40 nm above the TMCS surface, considerably more than observed for myosin subfragment 1 (S1; 6 nm). Together with the excellent actin transportation on TMCS, this strongly suggests that HMM adsorbs to TMCS mainly via its most C-terminal tail part. Consistent with this idea, fluorescence interference contrast (FLIC) microscopy showed that actin filaments are held by HMM 38 +/- 2 nm above the TMCS-surface with the catalytic site, on average, 20-30 nm above the surface. Viewed in a context with FLIC, QCM-D and TIRF results, the lack of actin motility and the limited actin binding on SiO(2) shows that HMM adsorbs largely via the actin-binding region on this surface with the C-terminal coiled-coil tails extending >50 nm into solution. The results and new insights from this study are of value, not only for the development of motor powered nanodevices but also for the interpretation of fundamental biophysical studies of actomyosin function and for the understanding of surface-protein interactions in general.
在体外运动分析中，肌动蛋白丝由表面吸附的肌球蛋白马达或更确切地说是肌球蛋白马达片段（如重酶解肌球蛋白，HMM）来推动。最近，人们已经在这一分析的基础上努力开发基于肌动球蛋白的纳米驱动设备，但这些进展受到对 HMM 吸附几何形状的有限理解的阻碍。因此，我们在这里研究 HMM 在三甲氯硅烷[TMCS]-衍生的疏水表面和亲水带负电荷的表面（SiO2）上的吸附几何形状。TMCS 表面在肌球蛋白和肌动蛋白的基础研究中非常重要，而这两种表面基质对马达驱动的纳米器件的发展都很重要。尽管 TMCS 和 SiO2 表面都几乎被 HMM 饱和（在 120μg mL-1下孵育），但在没有 ATP 的情况下，SiO2 上几乎没有肌动蛋白结合，而在有 ATP 的情况下也没有丝状滑动。这与 TMCS 上极好的肌动蛋白结合和运动形成鲜明对比。石英晶体微天平耗散（QCM-D）研究表明，HMM 层具有大量的蛋白质质量，在 TMCS 表面上方 40nm 处，明显高于肌球蛋白亚基 1（S1；6nm）。与 TMCS 上极好的肌动蛋白输送一起，这强烈表明 HMM 主要通过其最 C 末端的尾部部分吸附在 TMCS 上。荧光干涉对比（FLIC）显微镜显示，HMM 将肌动蛋白丝固定在 TMCS 表面上方 38 ± 2nm 处，催化部位平均在表面上方 20-30nm 处。从 FLIC、QCM-D 和 TIRF 结果的角度来看，SiO2 上肌动蛋白的运动性差和结合量有限表明，HMM 主要通过该表面上的肌动蛋白结合区域吸附，C 末端卷曲螺旋尾部延伸超过 50nm 进入溶液。这项研究的结果和新见解不仅对马达驱动纳米器件的开发具有价值，而且对肌球蛋白和肌动蛋白功能的基础生物物理研究的解释以及对表面-蛋白质相互作用的一般理解也具有价值。
