Just as humans use machines such as computers or cars to carry out complex tasks in day-to-day life, a cell uses many different kinds of machines to grow, to maintain its health, and to replicate. Unlike a computer or car, though, machines in the cell are often built only when needed for a particular task and are then disassembled; they are dynamic rather than static. An example of this is the machine that reads the information stored in DNA, the transcriptional machinery. More than fifty different proteins come together to form the transcriptional machinery at a specific gene and then the machinery is disassembled as the reading process begins. In part because they are short-lived, the interactions between proteins that comprise such machines are difficult to capture and visualize. This project focuses on developing novel tools such that dynamic machineries in the cell can be captured and studied in their native environment for the first time, tools that will be made available to the broader scientific community. The undergraduate students and graduate students working on this project will receive cross-disciplinary training in chemistry and biology and will share this knowledge in local Michigan K-8 classrooms.
With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Professor Anna Mapp of the University of Michigan to develop novel strategies for the discovery and characterization of transient, modest affinity protein-protein interactions in the native environment of the cell. Protein-protein interactions (PPIs) underlie all cellular process, serving to localize chemistries to specific cellular locations in a time-dependent fashion. The variety of PPIs in terms of affinity and surface is astonishing, with strengths ranging from picomolar to millimolar and surface areas from <500 Ã…2 to >3000 Ã…2. PPIs with high affinity and small surface areas most resemble protein-ligand interactions and they are the most amenable to characterization by standard genetic and biophysical methodologies; not surprisingly, they are the most well-defined in terms of function and mechanism. However, many cellular machineries rely upon modest affinity PPIs to accomplish their function. Traditional genetic, biochemical and biophysical strategies for characterizing such interactions are often ineffective and PPIs that fall within the low affinity range are the most poorly characterized in term of structure and mechanism in addition to having the smallest number of small molecule probes available. The tools developed through this project will enable time-dependent capture of such PPIs through the site-specific incorporation of photoactivatable functional groups into one or more protein binding partners. Additionally, a comparison of distinct photocrosslinking functional groups across a continuum of protein-protein interfaces will be used to build a framework for experimental design targeting any PPI of interest.
