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Methods. 2018 May 1;140-141:40-51. doi: 10.1016/j.ymeth.2018.02.002. Epub 2018 Feb 13.

Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy.

Author information

1
Department of Chemistry, University of Akron, Akron, OH 44325, USA.
2
Food Animal Health Research Program, Ohio Agriculture Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH 44691, USA.
3
Department of Biology, University of Akron, Akron, OH 44325, USA.
4
Department of Chemistry, University of Akron, Akron, OH 44325, USA. Electronic address: asmith5@uakron.edu.

Abstract

Fluorescence cross-correlation spectroscopy (FCCS) is an advanced fluorescence technique that can quantify protein-protein interactions in vivo. Due to the dynamic, heterogeneous nature of the membrane, special considerations must be made to interpret FCCS data accurately. In this study, we describe a method to quantify the oligomerization of membrane proteins tagged with two commonly used fluorescent probes, mCherry (mCH) and enhanced green (eGFP) fluorescent proteins. A mathematical model is described that relates the relative cross-correlation value (fc) to the degree of oligomerization. This treatment accounts for mismatch in the confocal volumes, combinatoric effects of using two fluorescent probes, and the presence of non-fluorescent probes. Using this model, we calculate a ladder of fc values which can be used to determine the oligomer state of membrane proteins from live-cell experimental data. Additionally, a probabilistic mathematical simulation is described to resolve the affinity of different dimeric and oligomeric protein controls.

KEYWORDS:

Dimerization; FRET; Fluorescence correlation spectroscopy; Fluorescence cross-correlation spectroscopy; Live cell fluorescence; Membrane protein dimerization

PMID:
29448037
PMCID:
PMC5999557
DOI:
10.1016/j.ymeth.2018.02.002
[Indexed for MEDLINE]
Free PMC Article

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