Physical Chemistry Seminar: Professor Alexander Barnes, Washington University in St. Louis (Host: Lynette Cegelski)
"Dynamic Nuclear Polarization, Electron Decoupling, and Magic Angle Spinning Spheres for HIV Cure Research"
About the Speaker
Alexander received his B.A. in Chemistry with Honors from Whitman college, and performed research at Washington University in St. Louis as an undergraduate researcher applying magic angle spinning (MAS) NMR to materials science with Sophia Hayes. Alexander than joined Robert Griffin’s group at MIT where he developed NMR probes, gyrotrons, waveguides, and cryogenics for high-field high-resolution DNP enhanced NMR. He then moved to Stanford University to work with Lynette Cegelski employing NMR to understand protein kinase C (PKC) modulation. Since 2012 Alexander has been an Assistant Professor in the Chemistry Department of Washington University in St. Louis. His group focuses on inventing instrumentation for MAS DNP, including frequency agile gyrotrons for pulsed DNP and electron decoupling and cryogenics to perform MAS < 6 Kelvin. His group leverages gains of >2000 fold in NMR sensitivity afforded from DNP to characterize the structure and dynamics of PKC modulation, both in vitro and within intact human cells. He is active in applying this molecular understanding of PKC activation to activate latent HIV reservoirs for HIV cure research.
NMR spectroscopy can elucidate atomic level structure and motion of chemical architectures ranging from the surface of materials to biomolecules within intact cells. Yet, NMR is plagued by poor sensitivity—solid state NMR signal averaging can last from minutes to months. In order to increase NMR sensitivity by more than factors of 50,000, our laboratory is inventing novel technology and developing new spin physics methodology. Critical to our efforts is the utilization of electron paramagnetic resonance (EPR) to transfer enhanced sensitivity from electrons to nuclei in a process known as dynamic nuclear polarization (DNP). Continuous wave (CW) DNP approaches at a sample temperature of 100 Kelvin can boost NMR signals by factors of 1000 in favorable samples. To extend on this now commercially available technology, we are developing pulsed DNP spectrometers (analogous to pulsed EPR and NMR) and cooling spinning samples to 4.2 Kelvin.[2,3,4] Pulsed EPR control in conjunction with magic angle spinning (MAS) also enables us to remove detrimental electron-nuclear spin interactions by decoupling the electron spins.
Utilizing the superb precision of solid state NMR structural measurements, we have resolved the conformational entropy present within an ensemble of (protein kinase C) PKC ligands. PKC is a drug target of latency reversal agents important to HIV cure research. The conformational entropy maps well onto the results of long (>500 microsecond) molecular dynamics trajectories. Advancements in DNP instrumentation and methodology are being applied to study the ligand binding pocket of PKC regulatory domains in vesicles. Whereas site specific 13C labeling of PKC domains affords resolution in cryogenic MAS DNP spectra in vitro, isotopic enrichment of ligands will extend these studies to binding characterization within intact human cells. Novel fluorescent DNP polarizing agents are being developed to determine the sub-cellular localization of PKC ligands for in-cell NMR and show targeted DNP enhancements in HEK293 cells. Lastly, in order to access magic angle spinning frequencies > 100 kHz , cool samples to temperatures <5 K with economical helium consumption rates, and improve microwave coupling, we have introduced spherical rotors for magic angle spinning.
1. Albert, Pahng, Alaniva, Sesti, Rand, Saliba, Scott, Choi, Barnes*. Instrumentation for Cryogenic Magic Angle Spinning Dynamic Nuclear Polarization Using 90 Liters of Liquid Nitrogen Per Day. Journal of Magnetic Resonance. 2017.
2. Hoff, Albert, Saliba, Scott, Choi, Mardini, Barnes*. Frequency Swept Microwaves for Hyperfine Decoupling and Time Domain Dynamic Nuclear Polarization. Solid State Nuclear Magnetic Resonance. 2015.
3. Sesti, Rand, Choi, Alaniva, Albert, Saliba, Scott, Barnes*. Magic Angle Spinning NMR at 4.2 Kelvin with Computational Fluid Dynamics Analysis of Fluid Flow and Temperature Gradients. Journal of Magnetic Resonance. 2017.
4. Scott, Saliba, Albert, Sesti, Gao, Golota, Alaniva, Choi, Sigurdsson, Barnes*. Frequency Agile Gyrotrons for Electron Decoupling and Pulsed Dynamic Nuclear Polarization. Journal of Magnetic Resonance. 2018.
5. Saliba, Sesti, Scott, Albert, Choi, Alaniva, Gao, Barnes*. Electron Decoupling with Dynamic Nuclear Polarization in Rotating Solids. Journal of the American Chemical Society. 2017.
6. Yang H., Staveness D., Ryckbosch S.M., Axtman A.D., Loy B.A., Barnes A.B., Pande V.S., Schaefer J., Wender P.A., Cegelski L. REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment. ACS Central Science, 4, 89-96. 2018.
7. Albert, Niu, Ramani, Marshall, Wender, Williams, Ratner, Barnes*, Kyei*. Combinations of Isoform-Targeted Histone Deacetylase Inhibitors and Bryostatin Analogues Display Remarkable Potency to Activate Latent HIV Without Global T-cell Activation. Nature Scientific Reports. 2017.
8. Albert B.J., Gao C., Sesti E.L., Saliba E.P., Alaniva N., Scott F.J., Barnes A.B.* Dynamic nuclear polarization NMR in human cells using fluorescent polarizing agents. Biochemistry. Accepted, ASAP. DOI: 10.1021/acs.biochem.8b00257. 2018.
9. Chen P.W., Albert B.J., Gao C, Alaniva N., Price L., Scott F. J., Saliba E.P., Sesti E.L., Fisher E.W., Barnes A.B.* Magic Angle Spinning Spheres. Science Advances. Accepted, in press. 2018.