Category Archives: bio NMR

Studies on the MxiH protein in T3SS needles using DNP-enhanced ssNMR spectroscopy #DNPNMR

Fricke, P., et al., Studies on the MxiH protein in T3SS needles using DNP-enhanced ssNMR spectroscopy. ChemPhysChem, 2014. 15(1): p. 57-60.

https://www.ncbi.nlm.nih.gov/pubmed/24282046

Bacterial T3SS needles formed by the protein MxiH are studied using DNP-enhanced ssNMR spectroscopy at 14.1 T (600 MHz). This technique provides spectra of good resolution, allowing us to draw conclusions about the protein dynamics. With the obtained signal enhancement, samples of limited quantity now get within reach of ssNMR studies.

Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization #DNPNMR

Viennet, T., et al., Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization. Angew Chem Int Ed Engl, 2016. 55(36): p. 10746-50.Viennet, T., et al., Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization. Angew Chem Int Ed Engl, 2016. 55(36): p. 10746-50.

https://www.ncbi.nlm.nih.gov/pubmed/27351143

Nuclear magnetic resonance (NMR) spectroscopy has the intrinsic capabilities to investigate proteins in native environments. In general, however, NMR relies on non-natural protein purity and concentration to increase the desired signal over the background. We here report on the efficient and specific hyperpolarization of low amounts of a target protein in a large isotope-labeled background by combining dynamic nuclear polarization (DNP) and the selectivity of protein interactions. Using a biradical-labeled ligand, we were able to direct the hyperpolarization to the protein of interest, maintaining comparable signal enhancement with about 400-fold less radicals than conventionally used. We could selectively filter out our target protein directly from crude cell lysate obtained from only 8 mL of fully isotope-enriched cell culture. Our approach offers effective means to study proteins with atomic resolution in increasingly native concentrations and environments.

[NMR] Postdoctoral Position in Biomolecular Solid-State NMR at USC, Los Angeles

From the Ampere Magnetic Resonance List

The Siemer lab at USC is looking for a postdoctoral associate with a background in NMR spectroscopy and knowledge of protein biochemical techniques. The lab studies the structure and dynamics of functional and toxic amyloid fibrils. To this goal we apply solid-state NMR spectroscopy in conjunction with other biochemical and biophysical methods.

The Siemer lab is part of the Protein Structure Center at USC and works in close collaboration wit the EPR lab of Ralf Langen and liquid-state NMR lab of Tobias Ulmer as part of an effort to investigate nervous system function with biophysical methods in an interdisciplinary environment.

The position is available immediately, and interested candidates should send their CV’s including the names of three references to Ansgar Siemer asiemer@usc.edu.

— 

Ansgar B Siemer 

Assistant Professor,

Biochemistry & Molecular Biology

Zilkha Neurogenetic Institute

Keck School of Medicine of USC

1501 San Pablo Street, ZNI 119F

Los Angeles, CA 90033

Tel: +1-323-442-2720

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This is the AMPERE MAGNETIC RESONANCE mailing list:

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NMR web database:

http://www.drorlist.com/nmr.html

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR #DNPNMR

Lange, S., et al., Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR. Sci Adv, 2016. 2(8): p. e1600379.

https://www.ncbi.nlm.nih.gov/pubmed/27551685

Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes #DNPNMR

Smith, A.N., et al., Molecular Rationale for Improved Dynamic Nuclear Polarization of Biomembranes. J Phys Chem B, 2016. 120(32): p. 7880-8.

https://www.ncbi.nlm.nih.gov/pubmed/27434371

Dynamic nuclear polarization (DNP) enhanced solid-state NMR can provide orders of magnitude in signal enhancement. One of the most important aspects of obtaining efficient DNP enhancements is the optimization of the paramagnetic polarization agents used. To date, the most utilized polarization agents are nitroxide biradicals. However, the efficiency of these polarization agents is diminished when used with samples other than small molecule model compounds. We recently demonstrated the effectiveness of nitroxide labeled lipids as polarization agents for lipids and a membrane embedded peptide. Here, we systematically characterize, via electron paramagnetic (EPR), the dynamics of and the dipolar couplings between nitroxide labeled lipids under conditions relevant to DNP applications. Complemented by DNP enhanced solid-state NMR measurements at 600 MHz/395 GHz, a molecular rationale for the efficiency of nitroxide labeled lipids as DNP polarization agents is developed. Specifically, optimal DNP enhancements are obtained when the nitroxide moiety is attached to the lipid choline headgroup and local nitroxide concentrations yield an average e(-)-e(-) dipolar coupling of 47 MHz. On the basis of these measurements, we propose a framework for development of DNP polarization agents optimal for membrane protein structure determination.

Structural biology applications of solid state MAS DNP NMR #DNPNMR

Akbey, U. and H. Oschkinat, Structural biology applications of solid state MAS DNP NMR. J Magn Reson, 2016. 269: p. 213-24.

http://www.ncbi.nlm.nih.gov/pubmed/27095695

Dynamic Nuclear Polarization (DNP) has long been an aim for increasing sensitivity of nuclear magnetic resonance (NMR) spectroscopy, delivering spectra in shorter experiment times or of smaller sample amounts. In recent years, it has been applied in magic angle spinning (MAS) solid-state NMR to a large range of samples, including biological macromolecules and functional materials. New research directions in structural biology can be envisaged by DNP, facilitating investigations on very large complexes or very heterogeneous samples. Here we present a summary of state of the art DNP MAS NMR spectroscopy and its applications to structural biology, discussing the technical challenges and factors affecting DNP performance.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies #DNPNMR

Gupta, R., et al., Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies. J Phys Chem B, 2016. 120(2): p. 329-39.

http://www.ncbi.nlm.nih.gov/pubmed/26709853

Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

Interview with Robert Tycko: On amyloids, Alzheimer disease, and solid-state NMR

Besides his contributions to solid-state NMR spectroscopy and it’s application to study bio-macromolecular systems, Rob Tycko is also very active in DNP research.

Skrynnikov, N.R. and R. Tycko, Interview with Robert Tycko: On amyloids, Alzheimer disease, and solid-state NMR. Concepts in Magnetic Resonance Part A, 2015. 44(4): p. 182-189.

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

Liao, S.Y., et al., Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR, 2016: p. 1-15.

http://www.ncbi.nlm.nih.gov/pubmed/26873390

Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~fourfold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105-160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 A from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

Dynamic Nuclear Polarization as an Enabling Technology for Solid State Nuclear Magnetic Resonance Spectroscopy

Smith, A.N. and J.R. Long, Dynamic Nuclear Polarization as an Enabling Technology for Solid State Nuclear Magnetic Resonance Spectroscopy. Analytical Chemistry, 2016. 88(1): p. 122-132.

http://dx.doi.org/10.1021/acs.analchem.5b04376

(This article does not seem to have an abstract, so I’m just posting the first paragraph here)

Magic angle spinning (MAS) solid state nuclear magnetic resonance spectroscopy (ssNMR) can yield unique and insightful information for complex systems; most notably structural and dynamical information can be obtained at atomic resolution.1−23 A particular strength of MAS ssNMR is it can be applied to heterogeneous systems, which are not amenable to study by other high-resolution experimental techniques due to sample characteristics. For example biomolecular assemblies, such as membrane proteins, are often not tractable for solubilization, a requisite for standard solution state NMR experiments, or crystallization, as needed for diffraction studies.1,24 Solid state materials with limited global order are often difficult to characterize by diffraction or other spectroscopic methods (e.g., UV− vis, IR, etc.), and information on reactive moieties, of particular interest for chemical and functional insight, can be difficult to differentiate from bulk signals.

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