Development of Solid State NMR Methods and Technology

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

Progress in FY2022 was in the following areas: (1) APPLICATION OF NEW RAPID MIXING/FREEZING TECHNOLOGY TO CALMODULIN/PEPTIDE COMPLEX FORMATION. In FY2022, we completed and published a study of the molecular mechanism by which calcium-loaded calmodulin forms a complex with a target peptide from myosin light chain kinase (M13 peptide). This collaborative study with the Clore group in LCP included a novel combination of pulsed EPR and solid state NMR measurements and took advantage of rapid mixing and rapid freeze-trapping technology developed in our laboratory over the past several years. Pulsed EPR, combined with double spin labeling of calmodulin, provided information about the time dependence of the calmodulin structure, while solid state NMR provided information about the time dependence of the M13 conformation and the time dependence of M13-calmodulin contacts. The combined data were well fit with a kinetic model, according to which two separate pathways lead to the final calmodulin/Ca2+/M13 complex. In one pathway, M13 binds first to the C-terminal domain of calmodulin, then proceeds to the final complex within about 0.5 ms. In the other pathway, M13 binds first to the N-terminal domain of calmodulin, then proceeds to an intermediate state in which calmodulin has undergone a conformational change but M13 is not yet fully ordered. This intermediate state proceeds to the final complex relatively slowly, in about 6 ms. This work was published in PNAS. It is the first example in which a biomolecular complex formation mechanism has been characterized by complementary time-resolved solid state NMR and EPR measurements and analyzed in terms of a specific kinetic model in which the various states have specific structural characteristics. (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. In FY2022, we completed a series of experiments on the bee venom peptide melittin, which converts from a largely monomeric state at 90 C to a tetrameric state at 30 C. Time-resolved solid state NMR data for evolution times in the 1-30 millisecond range demonstrated that the tetramerization process can be monitored after a rapid inverse temperature jump. Analyses of time-dependent 2D solid state NMR spectra showed that time scales for helix formation by melittin monomers and for the development of intermolecular contacts are nearly identical and can be fitted to a unidirectional dimerization model. Thus, helix formation and dimer formation occur as a concerted process, and tetramers form rapidly after dimerization. These results, including a full description of the novel inverse temperature-jump apparatus, were published in JACS. (3) 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). In FY2022, we completed a quantitative analysis of the experimental images, which indicates 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. A paper describing these results, including a full description of the novel low-temperature MRI apparatus, was published in PNAS. (4) NEW TRIRADICAL COMPOUNDS FOR DYNAMIC NUCLEAR POLARIZATION: We use dynamic nuclear polarization (DNP) to produce large signal enhancements in the time-resolved solid state NMR and low-temperature MRI experiments described above. DNP depends on addition of paramagnetic compounds to the NMR and MRI samples. In previous years, we showed that compounds containing three nitroxide free radical moieties are particularly favorable for DNP at very low temperatures (below 30 K). In FY2022, we synthesized and tested two new variants of these tri-nitroxide compounds, designed to have high solubilities over a wide range of pH values as required for various experiments in our lab. The synthesis methods and demonstrations of their effectiveness in DNP-enhanced solid state NMR at 25 K were published in the Journal of Magnetic Resonance.