Friday, March 27, 2015; 3:00 p.m.; Chem 200 (Kobilka Lecture Hall)
Department of Chemistry & Biochemistry Welcomes:
Dr. Joachim Mueller
Professor, Department of Physics and Astronomy, University of Minnesota Twin Cities
“Probing Peripheral Membrane Proteins inside the Living Cell with Fluorescence Fluctuation Microscopy”
Spatial and temporal fluctuations are inherent in every chemical system. These dynamic fluctuations are normally hidden from the observer, because most experimental techniques probe macroscopic samples, which results in an averaging out of fluctuations. However, by zooming into a small volume element, where the number of molecules of interest is small, fluctuations are ever-present. Two-photon fluorescence fluctuation spectroscopy provides an excellent method to observe these fluctuations with single molecule detection sensitivity. I will introduce the principles of fluorescence fluctuation spectroscopy and demonstrate how fluctuations encode the concentration, mobility, and stoichiometry of molecules with particular emphasis on fluorescently labeled proteins. An exciting application of two-photon fluorescence fluctuation spectroscopy is the study of protein interactions directly inside the living cell. I will discuss the challenges of performing fluctuation measurements in the intracellular environment, the progress we have made so far, and especially focus on applying fluorescence fluctuation spectroscopy to probe peripheral membrane proteins, which play an important role in cell signaling and other cellular events. Because these proteins only transiently associate with the membrane, they exist both in a soluble and membrane-bound state, which poses unique challenges for fluorescence fluctuation studies. We recently succeeded in addressing these challenges and developed methods that distinguish both protein populations. This development work has opened up new avenues for studying peripheral membrane proteins that were previously not possible inside the cell. I will demonstrate some of the new capabilities by quantifying protein-protein interactions and lipid binding of peripheral membrane proteins with fluorescence fluctuation microscopy.