Quantifying the Oligomeric States of Membrane Proteins in Cells through Super-Resolution Localizations.

Transitions between different oligomeric states of membrane proteins are essential for proper cellular functions. However, the quantification of their oligomeric states in cells is technically challenging. Here we developed a new method to quantify oligomeric state(s) of highly expressed membrane proteins using the probability density function of molecule density ( PDFMD) calculated from super-resolution localizations. We provided the theoretical model of PDFMD, discussed the effects of protein concentration, cell geometry, and photophysics of fluorescent proteins on PDFMD, and provided experimental criteria for proper quantification of oligomeric states. This method was further validated using simulated single-molecule fluorescent movies and applied to two membrane proteins, UhpT and SbmA in E. coli. The study shows that PDFMD is useful in quantifying oligomeric states of membrane proteins in cells that can help in understanding cellular tasks. Potential applications to proteins with higher oligomeric states under high concentration and limitations of our methodology were also discussed.