Brightness Characterization of Cellular and In Vitro Expressed Proteins

Abstract

Most biophysical characterizations of proteins and their interactions are performed in vitro. Because the cell interior is considerably more complex than the aqueous solution of in vitro experiments, differences in the strength and behavior of protein interactions in these two environments are anticipated. Characterization of such differences requires parallel studies of the same proteins and labels expressed in cells and in vitro. Cell-free expression systems offer a convenient method for comparing cell experiments with in vitro measurements. Because fluorescence fluctuation spectroscopy (FFS) determines the brightness of soluble proteins, it allows a direct comparison of protein interactions in the two systems. However, both environments present distinct obstacles to unbiased brightness measurement. The restricted geometry and size of the cellular environment influences the amplitude of the fluctuations. This effect is especially pronounced in thin cellular regions, where it leads to a biased brightness. We demonstrate that z-scan FFS abolishes this artifact for a thin slab geometry. We further extend the technique to multi-layer geometries that take differential protein localization into account. The main challenge for FFS experiments in the cell-free environment is the presence of fluorescent and non-fluorescent aggregates that are by-products of the expression reaction. Additionally, as cell-free experiments typically use small sample volumes, evaporation and surface-adsorption affect sample concentration. We introduce experimental protocols that limit these issues and permit unbiased FFS analysis. With these advances, we are able to successfully perform parallel brightness experiments on cellular and in vitro expressed proteins. This work was supported by a grant from the National Institutes of Health (GM64589).