Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations

• Published in: Biochimica et biophysica acta. General subjects Vol. 1868; no. 6; p. 130618
• Main Author: Clayton, Andrew H.A.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2024
• Subjects: biological control; Computer Simulation; energy transfer; fluorescence; Fluorescence lifetime imaging microscopy; Fluorescence Resonance Energy Transfer - methods; Forster resonance energy transfer; geometry; Humans; Image correlation spectroscopy; oligomerization; Photobleaching; Protein Multimerization; Protein Structure, Quaternary; Quaternary structure; spectroscopy; Image correlation spectroscopy; Fluorescence lifetime imaging microscopy; Forster resonance energy transfer; Quaternary structure
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2024.130618

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu. •Simulations reveal that combining donor photobleaching with lifetime-detected FRET enables FRET in dimers and in higher-order oligomers to be quantified.•The theoretical framework is described to enable quantitation of equilibria by image correlation analysis of lifetime-unmixed states during partial photobleaching. The new method is called pbFRET-FLIM-ICS.