We are currently making analogs of two proteins, soybean trypsin inhibitor and staphylococcal nuclease, that contain C13-enriched (80 times natural abundance) amino acids in specific sites. The C13-nmr peaks corresponding to the enriched residues will stand out from the background resonances, and resolution of single resonances should be possible in most cases. C13-enriched amino acids will be incorporated into soybean trypsin inhibitor by chemical and enzymatic reactions and into staphylococcal nuclease by microbiological procedures. We intend to investigate specific changes in the structure and side-chain mobility of these proteins that result from the binding of various ligands, including denaturants. Soybean trypsin inhibitor is one of the best- understood model systems for protein-protein interactions; it is highly resistant to denaturation. Staphylococcal nuclease binds nucleotides and metal ions; it can be unfolded and refolded reversibly under a variety of conditions. One of the goals of this research is to obtain information about the relative stability of various structural domains in proteins. Another goal is to investigate conformational equilibria (dynamic equilibria between multiple stable states) in these proteins. In this supplemental grant application we propose to extend the nmr studies of these proteins at 25.2 MHz for C13 (100 MHz for 1H) to also include 63.0 MHz for C13 (250 (MHz for 1H) by making use of the NIH- supported NMR Facility for Biomedical Studies at Carnegie-Mellon University. Our recent experience with this superconducting, cross- correlation spectrometer has demonstrated that the higher frequency will be of the utmost value in our research. First, the higher inherent sensitivity permits the use of smaller amounts of proteins and lower concentrations. Second, the higher resolution permits complete analysis of certain regions that cannot be interpreted at lower frequencies. Third, the wider range of shifts permits more accurate analysis of conformational changes in proteins. Fourth, studies of the frequency dependence of protein spectra permit the detection and quantitation of conformational equilibria in proteins.