Category Archives: Lipid Bilayers

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers #DNPNMR #NMR

Salnikov, E.S., et al., Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers. J Phys Chem B, 2015. 119(46): p. 14574-83. 

https://www.ncbi.nlm.nih.gov/pubmed/26487390

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h.

Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples #DNPNMR

Salnikov, E.S., et al., Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples. ChemPhysChem, 2017. 18(15): p. 2103-2113.

https://www.ncbi.nlm.nih.gov/pubmed/28574169

Dynamic nuclear polarization (DNP) boosts the sensitivity of NMR spectroscopy by orders of magnitude and makes investigations previously out of scope possible. For magic-angle-spinning (MAS) solid-state NMR spectroscopy studies, the samples are typically mixed with biradicals dissolved in a glass-forming solvent and are investigated at cryotemperatures. Herein, we present new biradical polarizing agents developed for matrix-free samples such as supported lipid bilayers, which are systems widely used for the investigation of membrane polypeptides of high biomedical importance. A series of 11 biradicals with different structures, geometries, and physicochemical properties were comprehensively tested for DNP performance in lipid bilayers, some of them developed specifically for DNP investigations of membranes. The membrane-anchored biradicals PyPol-C16, AMUPOL-cholesterol, and bTurea-C16 were found to exhibit improved g-tensor alignment, inter-radical distance, and dispersion. Consequently, these biradicals show the highest signal enhancement factors so far obtained for matrix-free membranes or other matrix-free samples and may potentially shorten NMR acquisition times by three orders of magnitude. Furthermore, the optimal biradical-to-lipid ratio, sample deuteration, and membrane lipid composition were determined under static and MAS conditions. To rationalize biradical performance better, DNP enhancement was measured by using the 13 C and 15 N signals of lipids and a peptide as a function of the biradical concentration, DNP build-up time, resonance line width, quenching effect, microwave power, and MAS frequency.

Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers

Salnikov, E.S., et al., Solid-State NMR/Dynamic Nuclear Polarization of Polypeptides in Planar Supported Lipid Bilayers. J Phys Chem B, 2015. 119(46): p. 14574-83.

http://www.ncbi.nlm.nih.gov/pubmed/26487390

Dynamic nuclear polarization has been developed to overcome the limitations of the inherently low signal intensity of NMR spectroscopy. This technique promises to be particularly useful for solid-state NMR spectroscopy where the signals are broadened over a larger frequency range and most investigations rely on recording low gamma nuclei. To extend the range of possible investigations, a triple-resonance flat-coil solid-state NMR probe is presented with microwave irradiation capacities allowing the investigation of static samples at temperatures of 100 K, including supported lipid bilayers. The probe performance allows for two-dimensional separated local field experiments with high-power Lee-Goldberg decoupling and cross-polarization under simultaneous irradiation from a gyrotron microwave generator. Efficient cooling of the sample turned out to be essential for best enhancements and line shape and necessitated the development of a dedicated cooling chamber. Furthermore, a new membrane-anchored biradical is presented, and the geometry of supported membranes was optimized not only for good membrane alignment, handling, stability, and filling factor of the coil but also for heat and microwave dissipation. Enhancement factors of 17-fold were obtained, and a two-dimensional PISEMA spectrum of a transmembrane helical peptide was obtained in less than 2 h.

Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR

Maciejko, J., et al., Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR. J Am Chem Soc, 2015. 137(28): p. 9032-43.

http://www.ncbi.nlm.nih.gov/pubmed/26102160

Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.

Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical

Fernandez-de-Alba, C., et al., Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical. Chemistry, 2015. 21(12): p. 4512-7.

http://www.ncbi.nlm.nih.gov/pubmed/25663569

Magic-angle spinning dynamic nuclear polarization (MAS-DNP) has been proven to be a powerful technique to enhance the sensitivity of solid-state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid-anchored polarizing agent. More specifically, we introduce a C16-functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo-protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix-free DNP approach that we recently introduced.

DNP-enhanced NMR on aligned Lipid Bilayers at ambient Temperature

Jakdetchai, O., et al., DNP-enhanced NMR on aligned Lipid Bilayers at ambient Temperature. J Am Chem Soc, 2014.

http://www.ncbi.nlm.nih.gov/pubmed/25333422

DNP-enhanced solid-state NMR has been shown to hold great potential for functional studies of membrane proteins at low temperatures due to its great sensitivity improvement. There are however numerous applications, for which experiments at ambient temperature are desirable and which would also benefit from DNP signal enhancement. Here, we demonstrate as a proof of concept that a significant signal increase for lipid bilayers under room temperature conditions can be achieved by utilizing the Overhauser effect. Experiments were carried out on aligned bilayers at 400 MHz/263 GHz using a stripline structure combined with a Fabry-Perot microwave resonator. A signal enhancement of protons of up to -10 was observed. Our results demonstrate that Overhauser DNP at high field pro-vides efficient polarization transfer within insoluble samples, which is driven by fast local molecular fluc-tuations. Furthermore, our experimental setup offers an attractive option for DNP-enhanced solid-state NMR on ordered membranes and provides a general perspective towards DNP at ambient temperatures.

Have a question?

If you have questions about our instrumentation or how we can help you, please contact us.