Development of Solid State NMR Methods and Technology

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Progress in FY2021 was in the following areas: (1) APPLICATION OF NEW RAPID MIXING/FREEZING TECHNOLOGY TO CALMODULIN/PEPTIDE COMPLEX FORMATION. In 2018, we perfected an apparatus for rapid mixing and freezing that enables studies of transient intermediates in structural conversion processes on the millisecond time scale. In 2019, we completed and published an initial application of this technology (Jeon et al., PNAS 2019), to the folding and tetramerization process of the bee venom peptide melittin following a rapid pH jump (from pH 3 to pH 7). In FY2020, initiated and published our initial studies of the formation of the complex between calmodulin and one of its target peptide from skeletal muscle myosin light chain kinase (M13 peptide), using the same technology. In these experiments, we mix a calmodulin solution with a solution containing M13 and calcium (1.5 ms mixing time), then rapidly freeze the mixed solution ( sub-millisecond freezing time) after a variable evolution period for complex formation. Solid state NMR spectra were recorded for samples with evolution periods between 1 ms and 27 ms. Complex formation strongly affects the positions and widths of crosspeaks in 2D solid state NMR spectra. Using M13 samples with isotopically labeled residues at selected positions, we find that the C-terminal half of M13 adopts a fully helical conformation more rapidly than the N-terminal half upon complex formation (2 ms vs. 10 ms). Contacts between M13 and methionine sidechains of calmodulin follow the same pattern, developing more rapidly for the C-terminal half of M13, which binds to the N-terminal half of calmodulin. In FY2021, we have extended our rapid mixing and freezing technology to pulsed EPR spectroscopy, in collaboration with the Clore lab in LCP. Pulsed EPR, combined with double spin labeling of calmodulin, provides information about the time dependence of the calmodulin structure with the same time resolution as our solid state NMR measurements. We have acquired a complete set of data (both solid state NMR and pulsed EPR) for the calmodulin/M13 system and are analyzing the data to determine the precise kinetic model that describes complex formation in the case of calmodulin that is preloaded with calcium. We expect to publish this work within the next several months. (2) INVERSE TEMPERATURE-JUMP NMR: As an alternative approach to rapid mixing for time-resolved solid state NMR, we have developed a simple method for changing the temperature of a protein solution from 90 C to 30 C in about 0.6 milliseconds. This method involves flowing the solution through copper capillary tubes that are soldered to hot and cold copper blocks. Initial experiments have been performed on melittin, which converts from a largely monomeric state at 90 C to a tetrameric state at 30 C. We have acquired time-resolved solid state NMR data for evolution times in the 1-30 millisecond range, verifying that the tetramerization process can be monitored after a rapid inverse temperature jump. We expect to publish this work within the next several months. (2) MICRON-SCALE MRI: Using low-temperature magnetic resonance imaging (MRI) and dynamic nuclear polarization (DNP) technology developed in our lab in recent years, we have obtained the first DNP-enhanced MRI images at very low temperatures (5 Kelvin), where the resulting enhancements of NMR signals can permit significant improvements in image resolution. Specifically, we have obtained images of a test sample, consisting of 9 micron-diameter glass beads immersed in glycerol/water within a 40 micron-inner-diameter capillary. The nominal resolution of these images is 1.7 microns (in all three spatial dimensions). Quantitative analyses of the experimental images indicate that the true resolution is approximately equal to the nominal resolution (definitely better than 2 micron isotropic resolution). This is a new world's record for resolution in MRI. We expect to publish this work within the next several months.