Wednesday, March 29, 2017

Quantitative analysis of molecular transport across liposomal bilayer by J-mediated 13C Overhauser dynamic nuclear polarization

Cheng, C.Y., O.J. Goor, and S. Han, Quantitative analysis of molecular transport across liposomal bilayer by J-mediated 13C Overhauser dynamic nuclear polarization. Anal Chem, 2012. 84(21): p. 8936-40.

We introduce a new NMR technique to dramatically enhance the solution-state (13)C NMR sensitivity and contrast at 0.35 T and at room temperature by actively transferring the spin polarization from Overhauser dynamic nuclear polarization (ODNP)-enhanced (1)H to (13)C nuclei through scalar (J) coupling, a method that we term J-mediated (13)C ODNP. We demonstrate the capability of this technique by quantifying the permeability of glycine across negatively charged liposomal bilayers composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The permeability coefficient of glycine across this DPPC/DPPG bilayer is measured to be (1.8 +/- 0.1) x 10(-11)m/s, in agreement with the literature value. We further observed that the presence of 20 mol % cholesterol within the DPPC/DPPG lipid membrane significantly retards the permeability of glycine by a factor of 4. These findings demonstrate that the high sensitivity and contrast of J-mediated (13)C ODNP affords the measurement of the permeation kinetics of small hydrophilic molecules across lipid bilayers, a quantity that is difficult to accurately measure with existing techniques.

Monday, March 27, 2017

The effect of Gd on trityl-based dynamic nuclear polarisation in solids

Ravera, E., et al., The effect of Gd on trityl-based dynamic nuclear polarisation in solids. Phys. Chem. Chem. Phys., 2015. 17(40): p. 26969-78.

In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the (13)C polarisation and decreases the (13)C polarisation buildup time of (13)C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a (13)C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an "effective electron-proton anisotropic hyperfine interaction parameter", and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.

Friday, March 24, 2017

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

Kim, Y., M. Liu, and C. Hilty, Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization. Anal Chem, 2016. 88(22): p. 11178-11183.

Protein-ligand interactions are frequently screened using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constant (KD) of a ligand of interest can be determined via a spin-spin relaxation measurement of a reporter ligand in a single scan when using hyperpolarization by means of dissolution dynamic nuclear polarization (D-DNP). Despite nearly instantaneous signal acquisition, a limitation of D-DNP for the screening of protein-ligand interactions is the required polarization time on the order of tens of minutes. Here, we introduce a multiplexed NMR experiment, where a single hyperpolarized ligand sample is rapidly mixed with protein injected into two flow cells. NMR detection is achieved simultaneously on both channels, resulting in a chemical shift resolved spin relaxation measurement. Spectral resolution allows the use of reference compounds for accurate quantification of concentrations. Simultaneous use of two concentration ratios between protein and ligand broadens the range of KD that is accurately measurable in a single experiment to at least an order of magnitude. In a comparison of inhibitors for the protein trypsin, the average KD values of benzamidine and benzylamine were found to be 12.6 +/- 1.4 muM and 207 +/- 22 muM from three measurements, based on KD = 142 muM assumed known for the reporter ligand 4-(trifluoromethyl)benzene-1-carboximidamide. Typical confidence ranges at 95% evaluated for single experiments were (8.3 muM, 20 muM) and (151 muM, 328 muM). The multiplexed detection of two or more hyperpolarized samples increases throughput of D-DNP by the same factor, improving the applicability to most multipoint measurements that would traditionally be achieved using titrations.

Wednesday, March 22, 2017

Perspectives for hyperpolarisation in compact NMR

Halse, M.E., Perspectives for hyperpolarisation in compact NMR. TrAC Trends in Analytical Chemistry, 2016. 83, Part A: p. 76-83.

Nuclear magnetic resonance (NMR) is one of the most powerful analytical techniques currently available, with applications in fields ranging from synthetic chemistry to clinical diagnosis. Due to the size and cost of high-field spectrometers, NMR is generally considered to be ill-suited for industrial environments and field work. This conventional wisdom is currently being challenged through the development of NMR systems that are smaller, cheaper, more robust and portable. Despite remarkable progress in this area, potential applications are often limited by low sensitivity. Hyperpolarisation techniques have the potential to overcome this limitation and revolutionise the use of compact NMR. This review describes the state-of-the-art in NMR hyperpolarisation and presents promising examples of its application to compact NMR. Both the benefits and challenges associated with the different hyperpolarisation approaches are discussed and applications where these technologies have the potential to make a significant impact are highlighted.

Monday, March 20, 2017

Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags #DNPNMR

Rogawski, R., et al., Dynamic Nuclear Polarization Signal Enhancement with High-Affinity Biradical Tags. The Journal of Physical Chemistry B, 2017. 121(6): p. 1169-1175.

Dynamic nuclear polarization is an emerging technique for sensitizing solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable biradicals, the polarization source for the cross effect mechanism, are typically codissolved at millimolar concentrations with proteins of interest. Here we describe the high-affinity biradical tag TMP-T, created by covalently linking trimethoprim, a nanomolar affinity ligand of dihydrofolate reductase (DHFR), to the biradical polarizing agent TOTAPOL. With TMP-T bound to DHFR, large enhancements of the protein spectrum are observed, comparable to when TOTAPOL is codissolved with the protein. In contrast to TOTAPOL, the tight binding TMP-T can be added stoichiometrically at radical concentrations orders of magnitude lower than in previously described preparations. Benefits of the reduced radical concentration include reduced spectral bleaching, reduced chemical perturbation of the sample, and the ability to selectively enhance signals for the protein of interest.

Friday, March 17, 2017

One-thousand-fold enhancement of high field liquid nuclear magnetic resonance signals at room temperature

Liu, G., et al., One-thousand-fold enhancement of high field liquid nuclear magnetic resonance signals at room temperature. Nat Chem, 2017. advance online publication.

Nuclear magnetic resonance (NMR) is a fundamental spectroscopic technique for the study of biological systems and materials, molecular imaging and the analysis of small molecules. It detects interactions at very low energies and is thus non-invasive and applicable to a variety of targets, including animals and humans. However, one of its most severe limitations is its low sensitivity, which stems from the small interaction energies involved. Here, we report that dynamic nuclear polarization in liquid solution and at room temperature can enhance the NMR signal of 13C nuclei by up to three orders of magnitude at magnetic fields of ∼3 T. The experiment can be repeated within seconds for signal averaging, without interfering with the sample magnetic homogeneity. The method is therefore compatible with the conditions required for high-resolution NMR. Enhancement of 13C signals on various organic compounds opens up new perspectives for dynamic nuclear polarization as a general tool to increase the sensitivity of liquid NMR.

Wednesday, March 15, 2017

Biosilica-Entrapped Enzymes Studied by Using Dynamic Nuclear-Polarization-Enhanced High-Field NMR Spectroscopy #DNPNMR

Ravera, E., et al., Biosilica-Entrapped Enzymes Studied by Using Dynamic Nuclear-Polarization-Enhanced High-Field NMR Spectroscopy. ChemPhysChem, 2015. 16(13): p. 2751-2754.

Enzymes are used as environmentally friendly catalysts in many industrial applications, and are frequently immobilized in a matrix to improve their chemical stability for long-term storage and reusability. Recently, it was shown that an atomic-level description of proteins immobilized in a biosilica matrix can be attained by examining their magic-angle spinning (MAS) NMR spectra. However, even though MAS NMR is an excellent tool for determining structure, it is severely hampered by sensitivity. In this work we provide the proof of principle that NMR characterization of biosilica-entrapped enzymes could be assisted by high-field dynamic nuclear polarization (DNP).