Short-Range Distance Measurement by Transition Metal Ion FRET

Research output: Chapter in Book/Report/Conference proceedingBook chapterResearchpeer-review

Measurement of atomic-scale conformational dynamics in proteins has proved a challenging endeavor, although these movements are pivotal for understanding the mechanisms behind protein function. Herein we describe a fluorescence-based method that enables the measurement of distances between specific domains within a protein and how it might change during protein function. The method is transition metal ion Förster resonance energy transfer (tmFRET) and builds on the principle that the fluorescence emission from a fluorophore can be quenched in a distance-dependent manner by a colored transition metal such as nickel (Ni2+), copper (Cu2+), or cobalt (Co2+). It can be applied to literally any protein where it is possible to perform site-specific incorporation of a fluorescent molecule. This chapter will explain the use and applications of tmFRET in detail using incorporation of the dye with cysteine chemistry on a purified protein sample.

Original languageEnglish
Title of host publicationBiophysics of membrane proteins
Number of pages13
PublisherHumana Press
Publication date2020
Publication statusPublished - 2020
SeriesMethods in Molecular Biology

    Research areas

  • Conformational dynamics, Cysteine chemistry, Fluorescence spectroscopy, Förster resonance energy transfer, Intramolecular distance measurements, Protein purification, Transition metals

ID: 270666372