FLUORESCENCE RESONANCE ENERGY-TRANSFER SPECTROSCOPY IS A RELIABLE RULER FOR MEASURING STRUCTURAL-CHANGES IN PROTEINS - DISPELLING THE PROBLEM OF THE UNKNOWN ORIENTATION FACTOR
FLUORESCENCE RESONANCE ENERGY-TRANSFER SPECTROSCOPY IS A RELIABLE RULER FOR MEASURING STRUCTURAL-CHANGES IN PROTEINS - DISPELLING THE PROBLEM OF THE UNKNOWN ORIENTATION FACTOR
复制标题
DOI：
10.1006/jsbi.1995.1042
复制
发表时间：
1995-09-01
影响因子：
3
通讯作者：
MOENS, PDJ
中科院分区：
文献类型：
--
作者：
DOSREMEDIOS, CG;MOENS, PDJ
荧光共振能量转移（FRET）光谱已被广泛用于在10-100埃的距离内“测量”分子内或分子间的尺寸，该范围非常适合于探测蛋白质结构。FRET光谱的分辨率远低于X射线衍射（XRD），但该技术弥补了这一缺陷，特别擅长测量蛋白质的结构变化。然而，通过FRET光谱测量的绝对距离仍然存在问题，因为似乎是一个不合理的假设，即FRET距离的计算假设探针能够进行自由，各向同性运动。这种不确定性可能归因于取向因子的未知值，但其他因素也可能是重要的。常识表明，共价结合到蛋白质的氨基酸侧链的大（300-500 Da）疏水性FRET探针不能经历真正的旋转自由。如果是这样的话，计算出的距离充其量是有缺陷的，最坏的情况是毫无意义的。在本文中，我们认为，方向参数不再是一个重要的问题，在确定的FRET使用肽和蛋白质的距离。此外，我们认为，FRET可能是一个很好的形式的光谱测试模型的F-肌动蛋白。(C)出版社：Academic Press
Fluorescence resonance energy transfer (FRET) spectroscopy has been widely used to ''measure'' dimensions either within or between molecules over distances of 10-100 Angstrom, a range that is well suited to probing protein structure. The resolution of FRET spectroscopy is substantially lower than X-ray diffraction (XRD) but the technique makes up for this deficit by being particularly good at measuring structural changes in proteins. However, absolute distances measured by FRET spectroscopy remain problematical because of what appears to be an unjustified assumption, namely that calculations of FRET distances assume that the probes are able to undergo free, isotropic motion. This uncertainty may be ascribed to an unknown value for the orientation factor, but other factors may also be important. Common sense suggests that a large (300-500 Da) hydrophobic FRET probe covalently bound to an amino acid side chain of a protein can not undergo true rotational freedom. If this is so, the calculated distances would at best be flawed and at worst be meaningless. In this paper we argue that the orientation parameter is no longer an important issue in the determination of distances determined by FRET using peptides and proteins. Furthermore, we suggest that FRET may be a good form of spectroscopy for testing models of F-actin. (C) 1995 Academic Press, Inc.