Corry Ben, Jayatilaka Dylan, Martinac Boris, Rigby Paul
School of Biomedical, Biomolecular and Chemical Sciences, The University of Western Australia, Crawley, Australia.
Biophys J. 2006 Aug 1;91(3):1032-45. doi: 10.1529/biophysj.106.080713. Epub 2006 May 12.
Orientational fluorophores have been a useful tool in physical chemistry, biochemistry, and more recently structural biology due to the polarized nature of the light they emit and that fact that energy can be transferred between them. We present a practical scheme in which measurements of the intensity of emitted fluorescence can be used to determine limits on the mean and distribution of orientation of the absorption transition moment of membrane-bound fluorophores. We demonstrate how information about the orientation of fluorophores can be used to calculate the orientation factor kappa(2) required for use in FRET spectroscopy. We illustrate the method using images of AlexaFluor probes bound to MscL mechanosensitive transmembrane channel proteins in spherical liposomes.
由于取向荧光团发射光的偏振特性以及能量可在它们之间转移这一事实，它们在物理化学、生物化学以及最近的结构生物学中一直是一种有用的工具。我们提出了一种实用方案，其中发射荧光强度的测量可用于确定膜结合荧光团吸收跃迁矩取向的均值和分布的限制。我们展示了如何利用荧光团取向信息来计算荧光共振能量转移（FRET）光谱中所需的取向因子κ(2)。我们使用与球形脂质体中MscL机械敏感跨膜通道蛋白结合的AlexaFluor探针的图像来说明该方法。
