This proposal outlines investigations of proteins and model systems with three innovation magnetic resonance techniques: high frequency dynamic nuclear polarization (DNP), 140 GHz electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). (1) High frequency CW DNP/MAS spectra of a protein have recently been observed. The experiments will be employed to enhance signal strengths in MAS NMR spectra of large proteins -- T4 lysozyme, bacteriorhodopsin, and ribonucleotide reductase (RNR) -- containing exogenous and endogenous paramagnetic centers such as TEMPO and tyrosyl radicals, respectively. The experiments utilize a 140 GHz gryotron oscillator as a microwave source, and the principal investigator will develop a low temperature helium recirculating system for use with DNP/MAS probes. Signal enhancements of ca. 100 have been observed, resulting in a factors of 10,000 decrease in acquisition time. (2) Pulsed DNP experiments based on Hartman-Hahn cross polarization are planned for single crystals samples as well as powders. (3) 140 GHz CW EPR ENDOR investigations are planned for five different systems: (a) a fluorinated inhibitor of ribonucleotide reductase (RNR); (b) the X-intermediate formed on combining Fe and the apoRNR; (c) thiyl radicals associated with adenosylcobalamin; (d) glycyl radicals in pyruvate formate lyase; (e) tyrosyl radicals in photosystem-II (4) 140 GHz pulsed EPR experiments will be used to study the frequency, concentration and temperature dependence of the electron spin lattice relaxation (T1e) in model systems for DNP experiments and in proteins. The data are useful for optimizing the rate and magnitude of polarization enhancements in determining structures of proteins containing free radicals and paramagnetic centers, and are inherently interesting.
