The Chemistry Core is concerned with the application of synthetic chemistry to the elucidation of the molecular basis of function for the classes of integral membrane proteins that are the subjects of the research investigations outlined in the Bridging and Pilot projects. The Core will provide state-of-the-art technology to introduce biophysical probes into a wide range of molecular entities including expressed proteins, semisynthetic proteins, chimeric proteins and peptides, for in vitro studies and also for in vivo studies in the natural cellular environment. The power of the synthetic approach is that we can design and synthesize protein molecules with novel properties that are finely tuned to the specific biological questions being asked and to the biophysical tools that will be used to address them. Existing methods, including total protein synthesis, expressed protein ligation, chemical tags, and unnatural amino acid mutagenesis are available for immediate implementation in the bridging projects. Moreover, we seek to pioneer the development and implementation of the next generation of these technologies incorporating spectroscopy probes that will provide for readouts of molecular motions with unprecedented versatility and precision. We will work closely with both the Computational Core and the Protein Production Core to use chemistry to strategically position these probes within expressed integral membrane proteins or synthetic proteins to provide the most informative picture of functional, conformational, and dynamic changes. Our efforts are motivated by a compelling need to design several classes of next generation spectroscopic probes to precisely dissect the role of conformational changes in membrane protein function. Our general methods for total and semi-synthesis are designed to label proteins in a non- or minimally-perturbing fashion with probe nuclei to act as local reporter groups. This will greatly expand the possibilities to explore, heretofore, unattainable features of protein dynamics using high-resolution nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopy. Additionally, chemical protein synthesis and semi-synthesis also enables the precise, site-specific introduction of fluorescent and EPR labels into complex protein systems to correlate dynamic properties with biological function. Next generation chemical tag probes with specialized properties for high-resolution (e.g. single molecule and super-resolution) imaging, and strategies for selectively labeling individual proteins in vivo using misacylated tRNAs, will be developed with the bridging projects in order to provide the tools needed for molecular resolution of membrane protein dynamics, even in living cells.