Observing Protein Interactions and Their Stoichiometry in Living Cells by Brightness Analysis of Fluorescence Fluctuation Experiments

School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA.
Methods in enzymology (Impact Factor: 2.09). 01/2010; 472:345-63. DOI: 10.1016/S0076-6879(10)72026-7
Source: PubMed


A single fluorescently labeled protein generates a short burst of light whenever it passes through a tiny observation volume created within a biological cell. The average amplitude of the burst is related to the stoichiometry of the fluorescently labeled protein complex. Fluorescence fluctuation spectroscopy quantifies the burst amplitude by introducing the brightness parameter. Brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells. Not all fluorescent proteins are suitable for brightness experiments. Here we discuss how brightness properties of the fluorophore influence brightness measurements and how to identify a well-behaved fluorescent protein. Protein interactions and stoichiometry are determined from a brightness titration. Experimental details of brightness titration measurements are described together with the necessary calibration and control experiments.

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    • "If each subunit is tagged with only a single fluorophore, the normalized brightness will reflect the number of subunits in a complex. This is called Brightness Analysis [19]. In addition, FCS can also determine the average amount of time a protein complex remains in the observation volume [17]; a value related to the lateral diffusion coefficient of the complex, itself a function of viscosity, mass and hydrodynamic volume (and hence the conformation of the complex). "
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    ABSTRACT: Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.
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