Molecular Biology and Biochemistry Department, Wesleyan University.
Molecular Biology and Biochemistry Department, Wesleyan University; Molecular Biophysics Program, Wesleyan University.
J Vis Exp. 2022 Mar 16(181). doi: 10.3791/63433.
Förster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.
Förster 共振能量转移（FRET）是一种成熟的荧光基础方法，用于成功测量体外生物分子之间以及细胞内的距离。在 FRET 中，通过荧光强度或寿命的变化来测量能量转移效率，其与两个荧光分子或标记物之间的距离有关。从这些距离确定动力学和构象变化只是该方法在生物系统中的一些应用示例。在某些条件下，这种方法可以通过提供有关动力学、灵活性和对结合表面的适应的信息，补充和增强现有的 X 射线晶体结构。我们通过鉴定结合位点或二聚体亚基的取向，描述了使用 FRET 和相关距离测定来阐明结构特性。通过明智地选择标记位点，并且通常采用多种标记策略，我们已经成功地将这些映射方法应用于确定蛋白质-DNA 复合物和 SecA-SecYEG 蛋白易位系统中的全局结构特性。在 SecA-SecYEG 系统中，我们使用 FRET 映射方法来鉴定前蛋白结合位点，并确定结合信号序列区域的局部构象。本研究概述了执行 FRET 映射研究的步骤，包括鉴定合适的标记位点、讨论可能的标记物，包括非天然氨基酸残基、标记程序、如何进行测量以及解释数据。
