用于生物分子结构测定的单分子水平高精度荧光共振能量转移
High Precision FRET at Single-molecule Level for Biomolecule Structure Determination.
作者信息
Ma Junyan, Yanez-Orozco Inna S, Rezaei Adariani Soheila, Dolino Drew, Jayaraman Vasanthi, Sanabria Hugo
机构信息
Department of Chemistry, Clemson University.
Department of Physics and Astronomy, Clemson University.
出版信息
J Vis Exp. 2017 May 13(123):55623. doi: 10.3791/55623.
Abstract
A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.
摘要
本文介绍了一种在多参数荧光检测（MFD）模式下，如何在单分子水平使用Förster共振能量转移（FRET）进行高精度染料间距离测量的方案。MFD可最大限度地利用荧光的所有“维度”，以减少光物理和实验假象，并能在刚性生物分子中以高达约1 Å的精度测量染料间距离。该方法用于识别N-甲基-D-天冬氨酸（NMDA）受体配体结合域的三种构象状态，以解释配体结合后受体的激活情况。将已知晶体结构与实验测量结果进行比较时，对于更具动态性的生物分子，二者在小于3 Å的范围内相符。收集一组覆盖生物分子整个维度的距离限制条件，将有可能提供动态生物分子的结构模型。
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