Imaging of fluorescence anisotropy during photoswitching provides a simple readout for protein self-association

Namrata Ojha, Kristin H. Rainey and George H. Patterson ()
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Namrata Ojha: National Institutes of Health
Kristin H. Rainey: National Institutes of Health
George H. Patterson: National Institutes of Health

Nature Communications, 2020, vol. 11, issue 1, 1-11

Abstract: Abstract Monitoring of protein oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements. Although donors and acceptors are typically fluorescent molecules with different spectra, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Here, we describe homo-FRET measurements by monitoring anisotropy changes in photoswitchable fluorescent proteins while photoswitching to the off state. These offer the capability to estimate anisotropy in the same specimen during homo-FRET as well as non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psAFRET) with a number of test chimeras and example oligomeric complexes inside living cells. We also present an equation derived from FRET and anisotropy equations which converts anisotropy changes into a factor we call delta r FRET (drFRET). This is analogous to an energy transfer efficiency and allows experiments performed on a given homo-FRET pair to be more easily compared across different optical configurations.

Date: 2020
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DOI: 10.1038/s41467-019-13843-6

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