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bioRxiv · 10.1101/477307

A general method to quantify ligand-driven oligomerization using single- or two-photon excitation microscopy

Stoneman, M. R.·Biener, G.·Ward, R. J.·Pediani, J. D.·Badu, D. N.·Popa, I. V.·Milligan, G.·Raicu, V.
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Abstract

Current technologies for probing membrane protein association and stability in cells are either very laborious or lack the bandwidth needed for fully quantitative analysis. Here we introduce a platform, termed one- or two-dimensional fluorescence intensity fluctuation spectrometry, for determining the identity, abundance, and stability of oligomers of differing sizes. The sensitivity of this approach is demonstrated by using monomers and oligomers of known sizes in both solutions and cell membranes. The analysis was extended to uncover the oligomeric states and their stability for both the epidermal growth factor receptor, a receptor tyrosine kinase, and the G protein-coupled secretin receptor. In both cases, agonist ligand binding shifted the equilibrium from monomers or dimers to rather large oligomers. Our method can be used in conjunction with a variety of light-based microscopy techniques, is several orders of magnitude faster than current approaches, and is scalable for high-throughput analyses.

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Stoneman, M. R., Biener, G., Ward, R. J., Pediani, J. D., Badu, D. N., Popa, I. V., Milligan, G., Raicu, V.. 2018-11-23. A general method to quantify ligand-driven oligomerization using single- or two-photon excitation microscopy. https://doi.org/10.1101/477307

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