A major challenge confronting biochemists is the ability to dissect, reconstruct, and manipulate each of the thousands of metastable protein complexes that exist in organisms. Structure/function analyses on protein signaling assemblies, membrane-bound transporters, and protein machines are limited by the fact that many only assemble and function properly in vivo. A crosslinking tool that works under native conditions, crosslinks with all amino acids, needs no chemical initiation, and results in predictable structural changes would revolutionize the methods for studying protein complexes. This work will determine if site-specific incorporation of photocrosslinking amino acids into proteins in vivo can overcome the current limitations for studying how the function of protein complexes relates to their assembly and structural changes. While this research focuses on photocrosslinking of bacterial chemoreceptors and dimeric model systems, it will define the experimental parameters for site-specific photocrosslinking amino acids so they will have broad application in biochemistry and biological materials.
Broader impacts: Within the curriculum, this project will be used to stress how the fundamental principals of chemistry and biology are combined to study the structure and function of complex biological systems. Introductory students will see how the basics of chemical reactions, kinetics and thermodynamics relate to future understanding and solving of biological problems. Conversely, biochemistry students will experience how the chemical basis of biological problems is instrumental in deciphering a biological process. This research connects biology and chemistry in the teaching and research laboratories, and trains undergraduates in experimental design, data interpretation, the scientific method, and in the use of specific biochemistry and organic chemistry techniques.
