This proposal outlines investigation of proteins and model systems with three innovative magnetic resonance techniques: high frequency dynamic nuclear polarization (DNP), time domain 140 GHz electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). The research plan is divided into four Specific Aims: (1) Development of CW and Pulsed DNP methods involving high Ble fields, the CROSS-effect, and electron-nuclear cross polarization. (2) Applications of DNP to biological problems include studies of alpha-spectrin SH3 crystals, oriented bacteriorhodopsin and Pfl bacteriophage, amyloid fibrils, and O-17 MQMAS. Each of these projects involves an area of magnetic resonance where the enhanced signal intensities available with DNP could have a significant impact. (3) Investigations of pulsed EPR and ENDOR investigations for four different systems: (a) the """"""""3- minute"""""""" radical observed in the E441Q mutant of ribonucleotide reductase (RNR); (b) a model for the X-intermediate formed on combining Fe and apoRNR, and the X-intermediate in the protein itself; (c) the tyrosyl radical in RNR from yeast; (d) study of a difluorinated inhibitor of RNR, gemcitabine, with both Class I and Class II RNR's. (4) Development of advanced pulsed EPR/ENDOR at 140 GHz. These include TRIPLE, TRAPS, DEER and MQENDOR with the goal of enhancing the versatility and resolution of methods available to examine protein free radicals.
