NMR Reveals the Conformational Changes of Cytochrome C upon Interaction with Cardiolipin

Conformational change of cytochrome c (cyt c) caused by interaction with cardiolipin (CL) is an important step during apoptosis, but the underlying mechanism is controversial. To comprehensively clarify the structural transformations of cyt c upon interaction with CL and avoid the unpredictable alias that might come from protein labeling or mutations, the conformation of purified yeast iso–1 cyt c with natural isotopic abundance in different contents of CL was measured by using NMR spectroscopy, in which the trimethylated group of the protein was used as a natural probe. The data demonstrate that cyt c has two partially unfolded conformations when interacted with CL: one with Fe–His33 coordination and the other with a penta–coordination heme. The Fe–His33 coordination conformation can be converted into a penta–coordination heme conformation in high content of CL. The structure of cyt c becomes partially unfolded with more exposed heme upon interaction with CL, suggesting that cyt c prefers a high peroxidase activity state in the mitochondria, which, in turn, makes CL easy to be oxidized, and causes the release of cyt c into the cytoplasm as a trigger in apoptosis.

Analyzing Ca-41 sample at 1E-15 abundance level with cold atom trap techniques

<p>On earth, Calcium-41 is produced as a cosmogenic isotope via neutron capture process, leaving a natural isotopic abundance of 10<sup>-15</sup> on earth surface. Calcium is also of vital importance for the metabolism of biological organisms. Consequently, analysis of the long lived radioactive isotope Calcium-41 is of great importance in geoscience, archeology and life sciences. The half-life of Calcium-41 is 1.03 x 10<sup>5</sup> years. It is a good candidate in dating rock and bone samples ranging from 50,000 to 1,000,000 years old.</p><p>The available techniques for trace analysis of Calcium-41 include accelerator mass spectrometry (AMS) and resonance ionization mass spectroscopy (RIMS). The detection limit of RIMS is on the level of 10<sup>-11 </sup>due to the interference of Potassium-41, which is difficult to remove from the sample. The analysis with high-energy AMS is more expensive than the table top apparatus, and it also faces similar problem as RIMS method.</p><p>We develop an atom trap trace analysis(ATTA) apparatus for Calcium-41 analysis to the sensitivity of 10<sup>-15</sup> abundance level by one hour of single atom counting. ATTA uses laser tuned at the resonant wavelength for a specific element and isotope to slow down and capture single atom by fluorescence radiation. It has a very high selectivity of element and isotope, which is more advantageous than AMS and RIMS to avoid isobar interference. ATTA has been used in analysis of Krypton-81, Argon-39 dating of the hydrological samples. This work on high sensitivity Calcium-41 analysis is very promising in dating the geochemical sample to determine the exposure ages of rocks or in cosmochemistry for investigations on terrestrial ages.</p>

Competing Transfer Pathways in Direct and Indirect Dynamic Nuclear Polarization MAS NMR Experiments on HIV-1 Capsid Assemblies: Implications for Sensitivity and Resolution

Dynamic nuclear polarization-enhanced (DNP) magic angle spinning (MAS) NMR of biological systems is a rapidly growing field. Large signal enhancements make the technique particularly attractive for signal-limited cases, such as studies of complex biological assemblies or at natural isotopic abundance. However, spectral resolution is considerably reduced compared to ambient-temperature non-DNP spectra. Herein, we report a systematic investigation into sensitivity and resolution of 1D and 2D 13C-detected DNP MAS NMR experiments on HIV-1 CA tubular assemblies. We show that the magnitude and sign of signal enhancement as well as the homogeneous line width are strongly dependent on the biradical concentration, the dominant polarization transfer pathway, and the enhancement buildup time. Our findings provide guidance for optimal choice of sample preparation and experimental conditions in DNP experiments.

Sequential assignment of NMR spectra of peptides at natural isotopic abundance with Zero and Ultra-Low Field-TOCSY

A novel method dubbed ZULF-TOCSY results from the combination of Zero and Ultra-Low Field (ZULF) with high-field, high-resolution NMR, leading to a generalization of the concept of total correlation spectroscopy...

Preparation and certification of natural and 82Se-labelled selenomethionine reference materials

Herein, we present the certification campaign for two new selenomethionine Certified Reference Materials: SENS-1, a high-purity powder of natural isotopic abundance, and SEES-1, a solution of selenium-82 labelled selenomethionine.

Development of Lock-in Based Overtone Modulated MARY Spectroscopy for Detection of Weak Magnetic Field Effects

Modulated magnetically altered reaction yield (ModMARY) spectroscopy is a derivative variant of fluorescence detected magnetic field effect measurement, where the applied magnetic field has both a constant and a modulated component. As in many derivative spectroscopy techniques, the signal to noise ratio scales with the magnitude of the modulation. High modulation amplitudes, however, distort the signal and can obscure small features of the measured spectrum. In order to detect weak magnetic field effects (including the low field effect) a balance of the two has to be found. In this work we look in depth at the origin of the distortion of the MARY signal by field modulation. We then present an overtone detection scheme, as well as a data analysis method which allows for correct fitting of both harmonic and overtone signals of the modulation broadened MARY data. This allows us to robustly reconstruct the underlying MARY curve at different modulation depths. To illustrate the usefulness of the technique, we show measurements and analysis of a well known magnetosensitive system of pyrene / 1,3-dicyanobenzene (Py/DCB). The measurements of first (h1) and second (h2) harmonic spectra are performed at different modulation depths for both natural isotopic abundance, and perdeuterated pyrene samples.<br>

Development of Lock-in Based Overtone Modulated MARY Spectroscopy for Detection of Weak Magnetic Field Effects

Modulated magnetically altered reaction yield (ModMARY) spectroscopy is a derivative variant of fluorescence detected magnetic field effect measurement, where the applied magnetic field has both a constant and a modulated component. As in many derivative spectroscopy techniques, the signal to noise ratio scales with the magnitude of the modulation. High modulation amplitudes, however, distort the signal and can obscure small features of the measured spectrum. In order to detect weak magnetic field effects (including the low field effect) a balance of the two has to be found. In this work we look in depth at the origin of the distortion of the MARY signal by field modulation. We then present an overtone detection scheme, as well as a data analysis method which allows for correct fitting of both harmonic and overtone signals of the modulation broadened MARY data. This allows us to robustly reconstruct the underlying MARY curve at different modulation depths. To illustrate the usefulness of the technique, we show measurements and analysis of a well known magnetosensitive system of pyrene / 1,3-dicyanobenzene (Py/DCB). The measurements of first (h1) and second (h2) harmonic spectra are performed at different modulation depths for both natural isotopic abundance, and perdeuterated pyrene samples.<br>

High-resolution ex vivo NMR spectroscopy of human Z α1-antitrypsin

Genetic mutations predispose the serine protease inhibitor α1-antitrypsin to misfolding and polymerisation within hepatocytes, causing liver disease and chronic obstructive pulmonary disease. This misfolding occurs via a transiently populated intermediate state, but our structural understanding of this process is limited by the instability of recombinant α1-antitrypsin variants in solution. Here we apply NMR spectroscopy to patient-derived samples of α1-antitrypsin at natural isotopic abundance to investigate the consequences of disease-causing mutations, and observe widespread chemical shift perturbations for methyl groups in Z AAT (E342K). By comparison with perturbations induced by binding of a small-molecule inhibitor of misfolding we conclude that they arise from rapid exchange between the native conformation and a well-populated intermediate state. The observation that this intermediate is stabilised by inhibitor binding suggests a paradoxical approach to the targeted treatment of protein misfolding disorders, wherein the stabilisation of disease-associated states provides selectivity while inhibiting further transitions along misfolding pathways.

UAV-based gas monitoring systems for the underpinning of urban, agricultural and industrial emission roadmaps – a methodological approach

&lt;p&gt;Currently sampling of the atmosphere for gas emission measurements involves building towers or hiring airplanes - capital-intensive methods. Easy access to unmanned aerial vehicles (UAV) has opened-up new opportunities for remote gas sampling. The project Iso-2-Drone aims to develop and produce a modular UAV-based gas monitoring system for emission measurements to substitute current technologies. A key feature of the UAV-attached gas sampler design was the ready-to-use nature of the system. This meant that the system was designed to mesh with commonly available equipment, using collection vessels which can be easily and immediately measured by common continuous flow - isotope ratio mass spectrometer (CF-IRMS) instrumentation. The target compounds comprise the three major natural greenhouse gases CH&lt;sub&gt;4&lt;/sub&gt;, CO&lt;sub&gt;2&lt;/sub&gt; and N&lt;sub&gt;2&lt;/sub&gt;O to be measured at natural isotopic abundance and ambient levels.&lt;/p&gt;&lt;p&gt;We use 20 mL headspace vials for CH&lt;sub&gt;4&lt;/sub&gt; and CO&lt;sub&gt;2&lt;/sub&gt; sampling. Vials can be conditioned on-sight with our sample preparation prototype using repeatedly evacuating and synthetic air refilling cycles to prevent ambient air contamination. On the UAV-attached sampler atmospheric air is sampled passively by pressure compensation of the vacuum. N&lt;sub&gt;2&lt;/sub&gt;O is sampled actively via adsorption tubes, filled with Molecular Sieve 5&amp;#197; and conditioned in the lab. Both a prototype device and two UAV-attached samplers have been designed, built and are currently tested.&lt;/p&gt;&lt;p&gt;The measurement setup in the lab comprises of two autosamplers, a purge &amp; trap system (VSP 4000, IMT Innovative Maschinentechnik GmbH) and a headspace sampler (CTC CombiPal, Chromtech GmbH) in order to switch from ppb range necessary for CH&lt;sub&gt;4 &lt;/sub&gt;and N&lt;sub&gt;2&lt;/sub&gt;O to a ppm range for CO&lt;sub&gt;2&lt;/sub&gt;. For CO&lt;sub&gt;2 &lt;/sub&gt;measurements the CTC injects 600 &amp;#181;l of sampled air to a Restek Micropacked Column (Shin Carbon ST 100/120, 2m x 1mm ID and 1/16&amp;#8221; OD) within a Thermo Scientific Trace GC Ultra heated up from 40&amp;#176;C to 110&amp;#176;C, maintained for 5 min, before heating up to 180&amp;#176;C by 12&amp;#176;C per minute. Thereby CO&lt;sub&gt;2&lt;/sub&gt; is properly separated from the potentially interfering N&lt;sub&gt;2&lt;/sub&gt;O. For CH&lt;sub&gt;4&lt;/sub&gt; the residual air sample is cryo-focused at -140&amp;#176;C in a HayeSep D filled trap, transferred to the GC and targeted with a Poraplot Q (30m x 0.32mm) held at 35&amp;#176;C. Using the similar GC method and autosampler N&lt;sub&gt;2&lt;/sub&gt;O is desorbed after switching the autosampler to thermal desorption mode. All three analytes pass an oxidation/reduction reactor (1030&amp;#176;C) before they are introduced into the IRMS (Thermo Scientific DeltaV Advantage) via a universal gas interface (Thermo Scientific Conflo IV). The IRMS continuously scans the intensity of the mass-to-charge ratios of mass 44, 45, 46 for CH&lt;sub&gt;4&lt;/sub&gt; and CO&lt;sub&gt;2&lt;/sub&gt; and 28, 29 for N&lt;sub&gt;2&lt;/sub&gt;0 converted to N&lt;sub&gt;2&lt;/sub&gt;. &amp;#948;&lt;sup&gt;13&lt;/sup&gt;C and &amp;#948;&lt;sup&gt;15&lt;/sup&gt;N are referenced against calibrated laboratory reference gases.&lt;/p&gt;&lt;p&gt;We are currently tuning the methods and testing the prototypes and will present the lasted results and open questions at the conference.&lt;/p&gt;