Expanding Number and Brightness to Determine the Oligomer Size of Membrane Proteins in Live Cells as a Function of Concentration

Abstract

The oligomer size of many membrane proteins plays a key role in their activity, and changes with increasing concentration and in the presence of ligands. However, many methods struggle to determine the exact oligomer size of complexes larger than dimers. Number and Brightness (N&B) is a fluorescence fluctuation method that is well suited to determine the oligomer size of complexes by comparing the molecular brightness of a monomer control (and ideally dimer, trimer, etc. controls) to the molecular brightness of a protein of interest. By also determining the two-dimensional concentration of the protein in the cell membrane and making measurements over a broad range of receptor concentrations, N&B could be used to create oligomerization curves. Care must be taken to account for cell movement and photobleaching, as small deviations can cause a large error in the measured molecular brightness, and the error is a function of concentration. In order to account for these complications, we have developed an approach which uses a combination of a hydrogel to inhibit cell movement and computational methods to correct for photobleaching and movement. Our preliminary results show differences in the oligomerization behavior of several receptor tyrosine kinases and co-receptors—including EGFR, TrkB, and NRP1—in live cells. Moreover, we directly show how ligand bound to membrane receptors affects their oligomerization state.