The dynamic assembly and disassembly of proteins into molecular complexes lies at the heart of countless biological processes ranging from cell-to-cell communication to the assembly of viral particles. However, almost everything known about the process of protein assembly into macromolecular machines comes from studying them in a test tube, isolated from the millions of other biological molecules present in cells. This project will develop new optical microscopy tools for studying the nanoscale organization of multiple dynamic protein interactions within single living cells. This work will take advantage of a process known as fluorescence resonance energy transfer (FRET) to detect the binding of two proteins. FRET allows observation of protein interactions through exchange of fluorescence energy between two fluorescent tags on the scale of five nanometers (~1/10,000,000th of an inch) and has become very popular in recent years to detect protein interactions. One of the major limitations of FRET, however, is that it has only been applied to study the interactions between two proteins at a time, thereby limiting its utility for studying complex molecular assemblies. This project will develop new methods and instrumentation to allow simultaneous detection of the interactions of four or more proteins in any combination. In addition, this instrument will be able to image where they occur within the cell with superior resolution to conventional light microscopes. This advance will be made by taking advantage of new methods for interfering patterns of light with the sample and then using computer reconstruction approaches to improve the subcellular resolution by about a factor of 2.5 over conventional microscopes. By extending FRET microscopy to the coincident detection of multiple molecular interactions while simultaneously improving the resolution limit of the microscope, this project will provide a highly versatile tool for studying molecular assemblies within living cells. The project will enhance the education and training of undergraduate and graduate students, as well as provide summer research opportunities for high school teachers as part of the institution's Masters in Chemical Education program. The visiting teachers will develop new teaching and laboratory materials for South Dakota high school students, a high fraction of which are Native American. The outcomes from this work will be accessible through a new website (www.fretmicroscopy.org/) and wiki describing the instrumentation, methods and kinds of molecular information that can be recovered from them. In addition, the software for performing the FRET calculations and image reconstructions will be distributed through this website.
