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.

Agency
National Institute of Health (NIH)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIADK029029-20
Application #
9357204
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Project Start
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Support Year
20
Fiscal Year
2016
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
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