Watanabe Kaori, Muhle-Goll Claudia, Kellermayer Miklós S Z, Labeit Siegfried, Granzier Henk
VCAPP, Washington State University, Pullman, Washington 99164-6520, USA.
J Struct Biol. 2002 Jan-Feb;137(1-2):248-58. doi: 10.1006/jsbi.2002.4458.
Titin is a giant elastic protein responsible for passive force generated by the stretched striated-muscle sarcomere. Passive force develops in titin's extensible region which consists of the PEVK segment in series with tandemly arranged immunoglobulin (Ig)-like domains. Here we studied the mechanics of tandem Ig segments from the differentially spliced (I65-70) and constitutive (I91-98) regions by using an atomic force microscope specialized for stretching single molecules. The mechanical stability of I65-70 domains was found to be different from that of I91-98 domains. In the range of stretch rates studied (0.05-1.00 microm/s) lower average domain unfolding forces for I65-70 were associated with a weaker stretch-rate dependence of the unfolding force, suggesting that the differences in the mechanical stabilities of the segments derive from differences in the zero force unfolding rate (K(0)(u)) and the characteristic distance (location of the barrier) along the unfolding reaction coordinate (DeltaX(u)). No effect of calcium was found on unfolding forces and persistence length of unfolded domains. To explore the structural basis of the differences in mechanical stabilities of the two fragment types, we compared the amino acid sequence of I65-70 domains with that of I91-98 domains and by using homology modeling analyzed how sequence variations may affect folding free energies. Simulations suggest that differences in domain stability are unlikely to be caused by variation in the number of hydrogen bonds between the force-bearing beta-strands at the domain's N- and C-termini. Rather, they may be due to differences in hydrophobic contacts and strand orientations.
肌联蛋白是一种巨大的弹性蛋白，负责由拉伸的横纹肌肌节产生的被动力。被动力在肌联蛋白的可伸展区域产生，该区域由与串联排列的免疫球蛋白（Ig）样结构域串联的PEVK片段组成。在这里，我们使用专门用于拉伸单分子的原子力显微镜研究了来自差异剪接（I65 - 70）和组成型（I91 - 98）区域的串联Ig片段的力学特性。发现I65 - 70结构域的机械稳定性与I91 - 98结构域不同。在所研究的拉伸速率范围内（0.05 - 1.00微米/秒），I65 - 70的平均结构域展开力较低，且展开力对拉伸速率的依赖性较弱，这表明这些片段机械稳定性的差异源于零力展开速率（K(0)(u)）以及沿着展开反应坐标的特征距离（势垒位置）（DeltaX(u)）的差异。未发现钙对展开力和展开结构域的持久长度有影响。为了探索这两种片段类型机械稳定性差异的结构基础，我们将I65 - 70结构域的氨基酸序列与I91 - 98结构域的进行了比较，并通过同源建模分析了序列变异如何影响折叠自由能。模拟结果表明，结构域稳定性的差异不太可能由结构域N端和C端承载力的β链之间氢键数量的变化引起。相反，它们可能是由于疏水接触和链取向的差异。
