Suda H, Sasaki Y C, Oishi N, Hiraoka N, Sutoh K
Department of Biological Science and Technology, Tokai University, 317 Nishino, Numazu, Shizuoka, 410-0321, Japan.
Biochem Biophys Res Commun. 1999 Aug 2;261(2):276-82. doi: 10.1006/bbrc.1999.1007.
Elasticity of a two-dimensionally arranged myosin subfragment-1 (S1) was measured by using a surface forces apparatus. To prepare a two-dimensionally arranged S1-monolayer on a functionalized silver surface, we used genetically engineered Dictyostelium S1 molecules. A highly reactive cysteine residue was fused to the COOH-terminus using the recombinant DNA method. On the other hand, the maleimide groups was self-assembled onto a silver surface. Then the mutant S1 molecules were chemically bound to the functionalized silver surface at its COOH-terminus. This arrangement technique was necessary in order to create a stable S1-monolayer by chemical bond formation onto the silver surface. The occupied area of the single S1 on the silver surface was about 110 nm(2). In the interaction between the S1-monolayer and mica surfaces in aqueous solution, a long-range attractive force was observed. The elastic constants (stiffness and Young's modulus) of myosin S1 were evaluated from force-distance profiles in aqueous solution, using the Hertz theory. We found that the stiffness (or spring constant) and Young's modulus of S1 in the absence of nucleotide are 4.4 +/- 1.0 pN/nm and 0.71 +/- 0.16 GPa, respectively.
利用表面力装置测量了二维排列的肌球蛋白亚片段-1（S1）的弹性。为了在功能化银表面制备二维排列的S1单层，我们使用了基因工程改造的盘基网柄菌S1分子。利用重组DNA方法将一个高反应性的半胱氨酸残基连接到COOH末端。另一方面，马来酰亚胺基团自组装到银表面。然后，突变型S1分子在其COOH末端化学结合到功能化银表面。这种排列技术对于通过在银表面形成化学键来创建稳定的S1单层是必要的。单个S1在银表面的占据面积约为110 nm²。在水溶液中S1单层与云母表面的相互作用中，观察到了长程吸引力。利用赫兹理论从水溶液中的力-距离曲线评估了肌球蛋白S1的弹性常数（刚度和杨氏模量）。我们发现，在不存在核苷酸的情况下，S1的刚度（或弹簧常数）和杨氏模量分别为4.4±1.0 pN/nm和0.71±0.16 GPa。
