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 cells. However, some proteins have both soluble (cytoplasmic) and membrane-bound fractions. In such conditions, coexcitation of the membrane-bound and cytoplasmic proteins makes standard FFS unsuitable. In this work, we apply z-scan FFS to characterize the intensity profile of proteins with both a cytoplasmic and membrane-bound population. Analysis of the intensity profile determines unequivocally the intensity contributions from each membrane layer and the cytoplasm, as well as, the length of cytoplasm layer. Experimentally, we choose Matrix-EGFP (the MA domain of HTLV-1 Gag) as our model system and apply a dual color z-scan technique. Cells are cotransfected with Matrix-EGFP and mCherry and their emitted signal is split into two different detection channels based on color. Simultaneous fitting of both channels with separate model geometries aids in correct determination of parameters. The use of the dual color technique is possible because we demonstrate a co-localization of the observation volume and agreement in the point spread function shape factors for both the red and green channels. We extend our work by applying z-scan FFS to determine the brightness (stoichiometry) of the cytoplasmic protein fraction. Yet determining the brightness of the membrane-bound protein is significantly more challenging because of slower diffusion times, inhomogeneity in protein distribution, and possible brightness bias due to geometry and orientation effects. Here, we investigate the potential of z-scan FFS to address these factors and measure the unbiased brightness of membrane proteins. This study provides insight into the fundamental considerations for correct application of FFS in the study of proteins with both cytoplasmic and membrane-bound fractions. This work is supported by NIH grant GM064589 and NIH grant GM075401.