Jun 14, 2019

The effect of drug binding on specific sites in transmembrane helices 4 and 6 of the ABC exporter MsbA studied by DNP-enhanced solid-state NMR #DNPNMR

Spadaccini, Roberta, Hundeep Kaur, Johanna Becker-Baldus, and Clemens Glaubitz. “The Effect of Drug Binding on Specific Sites in Transmembrane Helices 4 and 6 of the ABC Exporter MsbA Studied by DNP-Enhanced Solid-State NMR.” Biochimica et Biophysica Acta (BBA) - Biomembranes 1860, no. 4 (April 2018): 833–40.

MsbA, a homodimeric ABC exporter, translocates its native substrate lipid A as well as a range of smaller, amphiphilic substrates across the membrane. Magic angle sample spinning (MAS) NMR, in combination with dynamic nuclear polarization (DNP) for signal enhancement, has been used to probe two specific sites in transmembrane helices 4 and 6 of full length MsbA embedded in lipid bilayers. Significant chemical shift changes in both sites were observed in the vanadate-trapped state compared to apo state MsbA. The reduced spectral line width indicates a more confined conformational space upon trapping. In the presence of substrates Hoechst 33342 and daunorubicin, further chemical shift changes and line shape alterations mainly in TM6 in the vanadate trapped state were detected. These data illustrate the conformational response of MsbA towards the presence of drugs during the catalytic cycle.

Jun 12, 2019

13C → 1H transfer of light-induced hyperpolarization allows for selective detection of protons in frozen photosynthetic reaction center

Bielytskyi, Pavlo, Daniel Gräsing, Kaustubh R. Mote, Karthick Babu Sai Sankar Gupta, Shimon Vega, P.K. Madhu, A. Alia, and Jörg Matysik. “13C → 1H Transfer of Light-Induced Hyperpolarization Allows for Selective Detection of Protons in Frozen Photosynthetic Reaction Center.” Journal of Magnetic Resonance 293 (August 2018): 82–91.

In the present study, we exploit the light-induced hyperpolarization occurring on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photoCIDNP) effect to boost the NMR signal intensity of selected protons via inverse crosspolarization. Such hyperpolarization transfer is implemented into 1H-detected twodimensional 13C-1H correlation magic-angle-spinning (MAS) NMR experiment to study protons in frozen photosynthetic reaction centers (RCs). As a first trial, the performance of such an experiment is tested on selectively 13C labeled RCs from the purple bacteria of Rhodobacter sphaeroides. We observed response from the protons belonging to the photochemically active cofactors in their native protein environment. Such an approach is a potential heteronuclear spin-torch experiment which could be complementary to the classical heteronuclear correlation (HETCOR) experiments for mapping proton chemical shifts of photosynthetic cofactors and to understand the role of the proton pool around the electron donors in the electron transfer process occurring during photosynthesis.

Jun 10, 2019

Research Fellow in Membrane Protein Structural Biology #EPR

Are you an ambitious researcher looking for your next challenge? Do you have an established background in structural biology of membrane proteins? Are you interested in ion channels and mechanical sensing? Do you want to further your career in one of the UKs leading research intensive Universities?

Applications are invited to conduct research investigating the structure and function of mechanosensitive ion channels. These systems form pores in the cell membrane and allow the passage of molecules in response to membrane tension. 

A multi-disciplinary approach combining molecular/chemical (cloning, recombinant expression and purification), structural (CryoEM and PELDOR/DEER spectroscopy), functional (Electrophysiology) and computational (MD simulations) methods will be employed to investigate gating of these systems and characterise their unique states. CryoEM facilities in the Astbury Centre at University of Leeds are state-of-the-art, including 2 x Titan Krios 300keV electron microscopes. The project will also involve trips to the St Andrews and Manchester EPR facilities for PELDOR/DEER experiments.

We are seeking a highly motivated and talented individual interested in undertaking a challenging and exciting 3 year BBSRC-funded post in the laboratory of Dr Christos Pliotas. The project aims at obtaining and solving novel CryoEM structures of mechanosensitive ion channels in distinct conformational states. Dynamics will be interrogated by PELDOR/DEER and MD within lipid/native environment and function will be assessed by single molecule electrophysiology.

You should have a PhD (or close to completion) in Molecular/Structural Biology, Biochemistry, Biophysics or a closely allied discipline; with significant experience in expression, purification and structural biology of membrane proteins and ion channels.

To explore the post further or for any queries you may have, please contact: 

Dr Christos Pliotas, Lecturer in Integrative Membrane Biology

Location:Leeds - Main Campus
Faculty/Service: Faculty of Biological Sciences
School/Institute: School of Biomedical Sciences
Category: Research
Grade: Grade 7
Salary: £33,199 to £39,609 p.a.
Due to funding limitations it is unlikely an appointment will be made above £35,211 p.a.
Working Time: 100%
Post Type: Full Time
Contract Type: Fixed Term (3 years - external funding)
Release Date: Monday 03 June 2019
Closing Date: Sunday 14 July 2019
Reference: FBSBM1119

Variable magnet arrays to passively shim compact permanent-yoke magnets

Überrück, Till, and Bernhard Blümich. “Variable Magnet Arrays to Passively Shim Compact Permanent-Yoke Magnets.” Journal of Magnetic Resonance 298 (January 2019): 77–84.

C-shaped permanent magnets offer a compromise between sample accessability and field strength as well as homogeneity compared to single-sided devices or Halbach arrays. A new approach to passively shim C-shaped dipole magnets is presented. It relies on the magnet poles being constructed from a set of adjustable magnet elements. Two pole concepts are introduced, which allow the correction of the field profile and passively shim the magnet without the need of additonal pole shoes or shim pieces.

Jun 7, 2019

Daube, Diane, M. Vogel, Beatrix Suess, and Bjoern Corzilius. “Dynamic Nuclear Polarization on a Hybridized Hammerhead Ribozyme: An Explorative Study of RNA Folding and Direct DNP with a Paramagnetic Metal Ion Cofactor,” n.d., 43. 

While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal 13C/15N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional 13C–13C (PDSD) as well as 15N–13C (TEDOR) correlation spectroscopy under “conventional” DNP enhancement. This contact is indicative of the ribozyme’s functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg2+ co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal ion DNP if the diamagnetic Mg2+ is substituted by a paramagnetic Mn2

Jun 5, 2019

Direct dynamic nuclear polarization of 15N and 13C spins at 14.1 T using a trityl radical and magic angle spinning #DNPNMR

Wang, Xiaoling, Bethany G. Caulkins, Gwladys Riviere, Leonard J. Mueller, Frederic Mentink-Vigier, and Joanna R. Long. “Direct Dynamic Nuclear Polarization of 15N and 13C Spins at 14.1 T Using a Trityl Radical and Magic Angle Spinning.” Solid State Nuclear Magnetic Resonance, April 2019, S0926204019300177.

We investigate solid-state dynamic nuclear polarization of 13C and 15N nuclei using monoradical trityl OX063 as a polarizing agent in a magnetic field of 14.1 T with magic angle spinning at ~100 K. We monitored the field dependence of direct 13C and 15N polarization for frozen [13C, 15N] urea and achieved maximum absolute enhancement factors of 240 and 470, respectively. The field profiles are consistent with polarization of 15N spins via either the solid effect or the cross effect, and polarization of 13C spins via a combination of cross effect and solid effect. For microcrystalline, 15Nenriched tryptophan synthase sample containing trityl radical, a 1500-fold increase in 15N signal was observed under microwave irradiation. These results show the promise of trityl radicals and their derivatives for direct polarization of low gamma, spin-½ nuclei at high magnetic fields and suggest a novel approach for selectively polarizing specific moieties or for polarizing systems which have low levels of protonation.

Jun 3, 2019

Brute-force solvent suppression for DNP studies of powders at natural isotopic abundance #DNPNMR

Thureau, Pierre, Marie Juramy, Fabio Ziarelli, Stephane Viel, and Giulia Mollica. “Brute-Force Solvent Suppression for DNP Studies of Powders at Natural Isotopic Abundance.” Solid State Nuclear Magnetic Resonance 99 (July 2019): 15–19.

A method based on highly concentrated radical solutions is investigated for the suppression of the NMR signals arising from solvents that are usually used for dynamic nuclear polarization experiments. The presented method is suitable in the case of powders, which are impregnated with a radical-containing solution. It is also demonstrated that the intensity and the resolution of the signals due to the sample of interest is not affected by the high concentration of radicals. The method proposed here is therefore valuable when sensitivity is of the utmost importance, namely samples at natural isotopic abundance.