Progress in FY2016 was made in the following areas: (1) RAPID MIXING AND FREEZING TECHNOLOGY FOR SSNMR. We have designed microfluidic mixer and freeze-quench systems for millisecond-time-scale studies of protein folding, aggregation, and ligand binding processes by solid state NMR. As an initial test, we have examined the pH-dependent folding and tetramer assembly process of the bee venom peptide melittin, using DNP-enhanced solid state NMR measurements to determine the conformational state of the peptide in frozen solutions at low pH, at neutral pH, and within 5-10 ms of a pH jump from low to neutral. Initial data indicate that we will be able to probe intermediate states in this process. We have also designed a novel rapid temperature-jump apparatus, in which protein solution temperatures can be switched from 80 C to 20 C within several milliseconds, as the solutions flow through heated and cooled copper capillary tubes. Initial experiments on folding of villin HP35 verify the performance of this apparatus. (2) MRI MICROSCOPY. We have demonstrated that 3D images of test samples can be acquired with 5 micron isotropic resolution in the case of liquids and 8 micron isotropic resolution in the case of solids, using the microcoil-based MRI system described in our FY2014 report. These results are at room temperature. The liquid state performance is similar to the best results obtained to date by other groups. The solid state performance is substantially better than any previous results from other groups. This work is described in a paper that is under review at J. Magn. Reson. We are now testing the performance of our MRI apparatus at low temperatures (20 K or less), where we can further enhance NMR signals by dynamic nuclear polarization. Solid state MRI images with 3 micron resolution (without DNP) are currently being acquired.

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U.S. National Inst Diabetes/Digst/Kidney
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Thurber, Kent; Tycko, Robert (2016) Low-temperature dynamic nuclear polarization with helium-cooled samples and nitrogen-driven magic-angle spinning. J Magn Reson 264:99-106
Tycko, Robert (2015) On the problem of resonance assignments in solid state NMR of uniformly ¹⁵N,¹³C-labeled proteins. J Magn Reson 253:166-72
Thurber, Kent R; Tycko, Robert (2014) Perturbation of nuclear spin polarizations in solid state NMR of nitroxide-doped samples by magic-angle spinning without microwaves. J Chem Phys 140:184201
Potapov, Alexey; Yau, Wai-Ming; Tycko, Robert (2013) Dynamic nuclear polarization-enhanced 13C NMR spectroscopy of static biological solids. J Magn Reson 231:5-14
Tycko, Robert (2013) NMR at Low and Ultralow Temperatures. Acc Chem Res :
Thurber, Kent R; Tycko, Robert (2012) Theory for cross effect dynamic nuclear polarization under magic-angle spinning in solid state nuclear magnetic resonance: the importance of level crossings. J Chem Phys 137:084508
Hu, Kan-Nian; Qiang, Wei; Bermejo, Guillermo A et al. (2012) Restraints on backbone conformations in solid state NMR studies of uniformly labeled proteins from quantitative amide 15N-15N and carbonyl 13C-13C dipolar recoupling data. J Magn Reson 218:115-27
Hu, Kan-Nian; Qiang, Wei; Tycko, Robert (2011) A general Monte Carlo/simulated annealing algorithm for resonance assignment in NMR of uniformly labeled biopolymers. J Biomol NMR 50:267-76
Thurber, Kent R; Tycko, Robert (2009) Measurement of sample temperatures under magic-angle spinning from the chemical shift and spin-lattice relaxation rate of 79Br in KBr powder. J Magn Reson 196:84-7
Hu, Kan-Nian; Tycko, Robert (2009) Zero-quantum frequency-selective recoupling of homonuclear dipole-dipole interactions in solid state nuclear magnetic resonance. J Chem Phys 131:045101

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