Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC.
Fluorescence-based Methods for the Study of Protein-Protein Interactions Modulated by Ligand Binding
Background: The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomersbut that the oligomers also may modulate the receptor function provides a promising avenue in the area ofdrug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacywhile reducing side effects. In this regard, determining the various oligomeric configurations and geometricsub-states of a membrane receptor is of utmost importance. Methods: In this report, we have reviewed two techniques that have proven to be valuable in monitoring thequaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescenceintensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiencyare analyzed using theoretical models of various quaternary structures to determine the geometry andstoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensitiesare analyzed to reveal quantitative information on the size and stability of protein oligomers. Results: We demonstrate the application of these techniques to a number of different fluorescence-based studiesof cells expressing fluorescently labeled membrane receptors, both in the presence and absence of variousligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regardingthe quaternary structure receptors form, as well as FIF and FRET for determining the relative abundanceof different-sized oligomers when an equilibrium forms between such structures. Conclusion: FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,which are of great benefit to structure‐based drug design.
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- Award ID(s):
- 1919670
- NSF-PAR ID:
- 10295916
- Date Published:
- Journal Name:
- Current Pharmaceutical Design
- Volume:
- 26
- Issue:
- 44
- ISSN:
- 1381-6128
- Page Range / eLocation ID:
- 5668 to 5683
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Basic Protocol 1 : Preparation of live cells expressing protein constructsBasic Protocol 2 : Image acquisition and noise correctionBasic Protocol 3 : Drawing and segmenting regions of interestBasic Protocol 4 : Calculating the molecular brightness and concentration of individual image segmentsBasic Protocol 5 : Combining data subsets using a manual procedure (Optional)Alternate Protocol 1 : Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5)Basic Protocol 6 : Performing meta‐analysis of brightness spectrogramsAlternate Protocol 2 : Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6)Basic Protocol 7 : Spot extraction and analysis using a manual procedure or by writing a program (Optional)Alternate Protocol 3 : Automated spot extraction and analysis (Optional; alternative to Protocol 7)Support Protocol : Monomeric brightness determination -
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.2174/1381612826666201116120934
