Brightness Characterization of Cytoplasmic & Membrane-Bound Protein Mixtures by Z-Scan Fluorescence Fluctuation Spectroscopy

Abstract

Fluorescence fluctuation spectroscopy (FFS) has been employed to quantify the stoichiometry of soluble proteins in the nucleus and cytoplasm of mammalian cells. However, some proteins have both cytoplasmic and membrane-bound components which render traditional FFS methods inapplicable due to coexcitation of the spatially distributed protein mixture. In this work, we apply z-scan FFS to characterize the brightness of both the membrane-bound and cytoplasmic populations of a protein. Experimentally, we choose HRas-EGFP as our membrane-bound/cytoplasmic model system. We use a combination of stationary FFS measurements to determine the brightness of membrane-bound and cytoplasmic HRas-EGFP and z-scan intensity profiles to correct for brightness bias due to thin layer geometry and coexcitation of membrane and cytoplasmic layers. Our data show HRas-EGFP to be monomeric at both the upper and lower plasma membrane and also in the cytoplasm. We further examine the brightness statistics of proteins by comparing the experimental brightness distribution of a membrane-bound protein system with that of a soluble protein system. This work is extended by creating a dimeric membrane protein system in order to establish a model for calibrating brightness and stoichiometry. The z-scan FFS measurement technique along with a reliable monomer/dimer calibration system will be important for future membrane protein stoichiometry studies. This research was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (PHY 0346782).