The Photon Counting Histogram - A Fluorescence Fluctuation Technique For Studying Protein-Protein Interactions

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

The photon counting histogram (PCH) is a recently introduced technique that exploits fluorescence fluctuations to distinguish molecules by their intrinsic fluorescence brightness. For example, a homodimer A2 will appear twice as bright as its monomer A, if each protein A carries one fluorescent label. The main focus of this proposal is the development of dual-color PCH. Adding a second detection channel and splitting the photons between both channels according to color will increase the sensitivity and capability of the PCH technique tremendously. Dual-color PCH combines the benefit of separating photons of different molecules by color with the intrinsic ability of PCH to distinguish the brightness of molecules. To illustrate this point, consider two proteins A and B with differently colored labels. A mixture of their homodimers, heterodimers and monomers (A2, B2, AB, A and B) is resolvable by dual-color PCH. A single measurement will determine the brightness of each component together with their concentrations. Currently, no other technique promises to deliver comparable performance. The second goal of this proposal is the application of dual-color PCH to study subunit interactions of an oligomeric enzyme. The association and dissociation of individual subunits is directly observed by dual- color PCH analysis and allows the determination of protein assembly paths under varying external conditions. The existing theory of PCH for a single channel will be modified to describe dual-channel detection. The algorithms and software programs to analyze the data will be developed and the performance of the dual-channel detection system will be characterized on a two-photon microscope. The practical resolvability of a mixture by dual-color PCH is from an experimental point of view of crucial importance. This project will address and answer this question by analyzing the influence of the photon count statistics on dual-color PCH. Another important consideration is the emission spectrum of a fluorophore. Most pairs of fluorophores have overlapping emission spectra and a perfect separation of the two fluorophores by color is not possible. Optical filter combinations that maximize the signal-to-noise ratio of dual-color PCH for dyes with overlapping emission spectra will be determined and tested. The experimental characterization of dual-color PCH will start with binary mixtures and will continue with mixtures of increasing complexity to determine the capability of dual-color PCH. In addition, dual-color PCH with excitation at multiple wavelengths will be implemented to further increase the performance of PCH. The subunit interactions of phosphofructokinase (PFK) will be studied by dual-color PCH. Specifically, the subunit exchange between hybrid tetramers and the dissociation and association of the oligomeric protein will be examined. Individual subunits of PFK will carry specific fluorophores, which allow PCH to distinguish the subunit composition and population of each fraction by color and absolute brightness. It is important to stress that dual-color PCH will have applications well beyond the study of oligomeric enzymes. The impact of this new technique will be felt in many other biological research fields. The association of proteins to form complexes is a general concept encountered in most regulation processes of cells. Dual-color PCH can detect and identify protein association on the single molecule level. An important and pressing task of the post-genomic era is the identification of the function of a large number of unknown proteins. Dual-color PCH can assist in this task by mapping out their protein-protein interactions.