Short-Range Distance Measurement by Transition Metal Ion FRET
Research output: Chapter in Book/Report/Conference proceeding › Book chapter › Research › peer-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 language | English |
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Title of host publication | Biophysics of membrane proteins |
Number of pages | 13 |
Publisher | Humana Press |
Publication date | 2020 |
Pages | 299-311 |
DOIs | |
Publication status | Published - 2020 |
Series | Methods in Molecular Biology |
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Volume | 2168 |
ISSN | 1064-3745 |
- Conformational dynamics, Cysteine chemistry, Fluorescence spectroscopy, Förster resonance energy transfer, Intramolecular distance measurements, Protein purification, Transition metals
Research areas
ID: 270666372