Heidecker M, Yan-Marriott Y, Marriott G
Biomolecular and Cellular Dynamics Research Group, Max Planck Institute for Biochemistry, Martinsried, Germany.
Biochemistry. 1995 Sep 5;34(35):11017-25. doi: 10.1021/bi00035a007.
Distance relationships between phalloidin binding sites on F-actin have been investigated using fluorescence resonance energy transfer (FRET) techniques in solution and on single F-actin filaments bound to heavy meromyosin (HMM). Filaments saturated with an equimolar concentration of fluoresceinisothiocyanatophalloidin (FITC-ph) as the donor and tetramethylrhodamineisothiocyanatophalloidin (TRITC-ph) as the acceptor and control filaments saturated with either FITC-ph or TRITC-ph were characterized by absorption and fluorescence spectroscopy and the in vitro motility assay. Fluorescence excitation polarization spectroscopy showed hetero-FRET occurred within colabeled filaments, whereas homo-FRET was observed in control filaments. The distance measured between adjacent phalloidin binding sites using randomly labeled FITC-ph and TRITC-ph was 37.2 A using steady-state spectral analysis and 36.9 A using time-resolved spectroscopy with a radial coordinate of 14.5 A. Measurements of the distance between fluorescent phalloidin groups using the atomic model of F-actin [Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826-836] suggest transfer occurs between adjacent phalloidin molecules on opposite actin helices (39 A), rather than between adjacent phalloidins along the same actin helix (55.4 A). A quantitative fluorescence microscope technique was described that measures the proximity of adjacent FITC-ph and TRITC-ph on single filaments immobilized on HMM. Here a distance of 36.2 A was calculated which was unchanged during ATP-dependent sliding of F-actin on HMM. Spatially resolved FRET measurements are being used to observe the effects of actin binding proteins on the structural properties along the length of single actin filaments.
利用溶液中的荧光共振能量转移（FRET）技术以及结合了重酶解肌球蛋白（HMM）的单根F-肌动蛋白丝，研究了F-肌动蛋白上鬼笔环肽结合位点之间的距离关系。用等摩尔浓度的异硫氰酸荧光素鬼笔环肽（FITC-ph）作为供体、四甲基罗丹明异硫氰酸酯鬼笔环肽（TRITC-ph）作为受体饱和的肌动蛋白丝，以及用FITC-ph或TRITC-ph饱和的对照肌动蛋白丝，通过吸收光谱、荧光光谱和体外运动分析进行表征。荧光激发偏振光谱显示，在共标记的肌动蛋白丝中发生了异源FRET，而在对照肌动蛋白丝中观察到了同源FRET。使用随机标记的FITC-ph和TRITC-ph，通过稳态光谱分析测得相邻鬼笔环肽结合位点之间的距离为37.2 Å，通过时间分辨光谱测得的距离为36.9 Å，径向坐标为14.5 Å。利用F-肌动蛋白的原子模型[Lorenz, M., Popp, D., & Holmes, K. C. (1993) J. Mol. Biol. 234, 826 - 836]测量荧光鬼笔环肽基团之间的距离，结果表明转移发生在相反肌动蛋白螺旋上相邻的鬼笔环肽分子之间（39 Å），而不是沿着同一肌动蛋白螺旋上相邻的鬼笔环肽之间（55.4 Å）。描述了一种定量荧光显微镜技术，用于测量固定在HMM上的单根肌动蛋白丝上相邻FITC-ph和TRITC-ph的距离。在此计算出的距离为36.2 Å，在F-肌动蛋白在HMM上进行ATP依赖性滑动过程中该距离保持不变。空间分辨FRET测量正被用于观察肌动蛋白结合蛋白对单根肌动蛋白丝长度方向结构特性的影响。
