β-Lactamases (EC 3.5.2.6) confer resistance against β-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against β-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative β-lactamase activity, sulbactam binding (a β-lactam analogue) in the low μM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known β-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its β-lactamase activity. Current study is the first report on β-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for β-lactamase activity.
- MeSH
- beta-laktamasy * chemie metabolismus MeSH
- Chlamydomonas reinhardtii * enzymologie MeSH
- magnetická rezonanční spektroskopie metody MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- rostlinné proteiny chemie metabolismus MeSH
- sekvence aminokyselin MeSH
- simulace molekulární dynamiky MeSH
- sulbaktam chemie farmakologie MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
The understanding and correct description of intermolecular hydrogen bonds are crucial in the field of multicomponent pharmaceutical solids, such as salts and cocrystals. Solid isonicotinic acid can serve as a suitable model for the development of methods that can accurately characterize these hydrogen bonds. Experimental solid-state NMR has revealed a remarkable temperature dependence and deuterium-isotope-induced changes of the chemical shifts of the atoms involved in the intermolecular hydrogen bond; these NMR data are related to changes of the average position of the hydrogen atom. These changes of NMR parameters were interpreted using periodic DFT path-integral molecular dynamics (PIMD) simulations. The small size of the unit cell of isonicotinic acid allowed for PIMD simulations with the computationally demanding hybrid DFT functional. Calculations of NMR parameters based on the hybrid-functional PIMD simulations are in excellent agreement with experiment. It is thus demonstrated that an accurate characterization of intermolecular hydrogen bonds can be achieved by a combination of NMR experiments and advanced computations.
- MeSH
- kyseliny isonikotinové * MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
Conventional Nuclear Magnetic Resonance (NMR) analysis relies on H-H/C-H interactions. However, these interactions are sometimes insufficient for an accurate and precise NMR analysis. In this study, we show that 31P NMR parameters can provide critical structural insights into the stereochemistry of phosphorus-containing compounds. For this purpose, we prepared a set of model phosphorus-based proline derivatives, separated diastereoisomers, and determined their absolute configuration by single-crystal X-ray diffraction. After supplementing these results by electronic circular dichroism (ECD) spectroscopy, we combined experimental data and DFT calculations from our model compounds to perform a detailed conformational analysis, thereby determining their relative configuration. Overall, our findings establish an experimental paradigm for combining 31P NMR spectroscopy with other optical methods to facilitate the stereochemical analysis of phosphorus-containing compounds.
The molecule of 2,2-dinitroethene-1,1-diamine (FOX-7) is one of the most interesting molecules with multiple redox centres stabilized by push-pull effect. To reveal the detailed mechanism of its electrochemical process radical intermediates formed in the course of its electrochemical reduction in organic aprotic media have been studied by in situ simultaneous electrochemical ESR measurements (SEESR). The radical generated on the second reduction step possesses an alternating line-width (AL) effect in the ESR spectra as a result of intramolecular dynamic processes in the timescale of ESR splitting constants. The spectra measured at different temperatures (230-335 K) were analysed with the help of a fitting program which includes a molecular dynamics. Observed dynamics describes well an asymmetric 2-site exchange model for the whole temperature range. With help of the optimized parameters and quantum chemical calculations this radical has been identified as 2,2-dinitroethane-1-amine-1-imine radical dianion, [(H2N)(HN)C=C(NO2)2]2-. The dynamic process responsible for the AL effect consists of mutual turning (changing of dihedral angle) of the both nitro groups, resulting in an intramolecular spin-density (electron) transfer. The dynamic parameters of the process have been established.
- Publikační typ
- časopisecké články MeSH
Electron paramagnetic resonance (EPR) spectroscopy represents an established tool to study properties of microenvironments, e.g. to investigate the structure and dynamics of biological and artificial membranes. In this study, the partitioning of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in ex vivo human abdominal and breast skin, ex vivo porcine abdominal and ear skin as well as normal and inflammatory in vitro skin equivalents was investigated by EPR spectroscopy. Furthermore, the stratum corneum (SC) lipid composition (as determined by high-performance thin-layer chromatography), SC lipid chain order (probed by infrared spectroscopy) and the SC thickness (investigated by histology) were determined in the skin models. X-band EPR measurements have shown that TEMPO partitions in the lipophilic and hydrophilic microenvironment in varying ratios in different ex vivo and in vitro skin models. Ex vivo human abdominal skin exhibited the highest amount of TEMPO in the lipophilic microenvironment. In contrast, the lowest amount of TEMPO in the lipophilic microenvironment was determined in ex vivo human breast skin and the inflammatory in vitro skin equivalents. Individual EPR spectra of epidermis including SC and dermis indicated that the lipophilic microenvironment of TEMPO mainly corresponds to the most lipophilic part of the epidermis, the SC. The amount of TEMPO in the lipophilic microenvironment was independent of the SC lipid composition and the SC lipid chain order but correlated with the SC thickness. In conclusion, EPR spectroscopy could be a novel technique to determine differences in the SC thickness, thus suitably complementing existing methods.
- MeSH
- břicho MeSH
- buněčné mikroprostředí MeSH
- chromatografie na tenké vrstvě MeSH
- cyklické N-oxidy chemie MeSH
- dospělí MeSH
- elektronová paramagnetická rezonance MeSH
- epidermis chemie MeSH
- kůže chemie cytologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- lipidy chemie MeSH
- mladý dospělý MeSH
- prasata MeSH
- prsy MeSH
- senioři MeSH
- spektrofotometrie infračervená MeSH
- spinové značení MeSH
- tloušťka kožní řasy MeSH
- zevní ucho MeSH
- zvířata MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Three-dimensional topological insulators are an important class of modern materials, and a strong spin-orbit coupling is involved in making the bulk electronic states very different from those near the surface. Bi2Se3 is a model compound, and 209Bi NMR is employed here to investigate the bulk properties of the material with focus on the quadrupole splitting. It will be shown that this splitting measures the energy band inversion induced by spin-orbit coupling in quantitative agreement with first-principle calculations. Furthermore, this quadrupole interaction is very unusual as it can show essentially no angular dependence, e.g., even at the magic angle the first-order splitting remains. Therefore, it is proposed that the magnetic field direction is involved in setting the quantization axis for the electrons, and that their life time leads to a new electronically driven relaxation mechanism, in particular for quadrupolar nuclei like 209Bi. While a quantitative understanding of these effects cannot be given, the results implicate that NMR can become a powerful tool for the investigation of such systems.
- Publikační typ
- časopisecké články MeSH
A flexible transceiver array based on transmission line resonators (TLRs) combining the advantages of coil arrays with the possibility of form-fitting targeting cardiac MRI at 7 T is presented. The design contains 12 elements which are fabricated on a flexible substrate with rigid PCBs attached on the center of each element to place the interface components, i.e. transmit/receive (T/R) switch, power splitter, pre-amplifier and capacitive tuning/matching circuitry. The mutual coupling between elements is cancelled using a decoupling ring-based technique. The performance of the developed array is evaluated by 3D electromagnetic simulations, bench tests, and MR measurements using phantoms. Efficient inter-element decoupling is demonstrated in flat configuration on a box-shaped phantom (Sij < -19 dB), and bent on a human torso phantom (Sij < -16 dB). Acceleration factors up to 3 can be employed in bent configuration with reasonable g-factors (<1.7) in an ROI at the position of the heart. The array enables geometrical conformity to bodies within a large range of size and shape and is compatible with parallel imaging and parallel transmission techniques.
- MeSH
- elektromagnetická pole MeSH
- fantomy radiodiagnostické MeSH
- lidé MeSH
- magnetická rezonanční tomografie přístrojové vybavení MeSH
- počítačová simulace MeSH
- poměr signál - šum MeSH
- rádiové vlny MeSH
- srdce diagnostické zobrazování MeSH
- trup diagnostické zobrazování MeSH
- vylepšení obrazu MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Standard spectral density mapping protocols, well suited for the analysis of (15)N relaxation rates, introduce significant systematic errors when applied to (13)C relaxation data, especially if the dynamics is dominated by motions with short correlation times (small molecules, dynamic residues of macromolecules). A possibility to improve the accuracy by employing cross-correlated relaxation rates and on measurements taken at several magnetic fields has been examined. A suite of protocols for analyzing such data has been developed and their performance tested. Applicability of the proposed protocols is documented in two case studies, spectral density mapping of a uniformly labeled RNA hairpin and of a selectively labeled disaccharide exhibiting highly anisotropic tumbling. Combination of auto- and cross-correlated relaxation data acquired at three magnetic fields was applied in the former case in order to separate effects of fast motions and conformational or chemical exchange. An approach using auto-correlated relaxation rates acquired at five magnetic fields, applicable to anisotropically moving molecules, was used in the latter case. The results were compared with a more advanced analysis of data obtained by interpolation of auto-correlated relaxation rates measured at seven magnetic fields, and with the spectral density mapping of cross-correlated relaxation rates. The results showed that sufficiently accurate values of auto- and cross-correlated spectral density functions at zero and (13)C frequencies can be obtained from data acquired at three magnetic fields for uniformly (13)C-labeled molecules with a moderate anisotropy of the rotational diffusion tensor. Analysis of auto-correlated relaxation rates at five magnetic fields represents an alternative for molecules undergoing highly anisotropic motions.
- MeSH
- algoritmy * MeSH
- interpretace statistických dat * MeSH
- magnetická rezonanční spektroskopie s uhlíkem 13C metody MeSH
- magnetické pole MeSH
- malá interferující RNA analýza chemie MeSH
- počítačové zpracování signálu * MeSH
- reprodukovatelnost výsledků MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Proteins, which, in their native conditions, sample a multitude of distinct conformational states characterized by high spatiotemporal heterogeneity, most often termed as intrinsically disordered proteins (IDPs), have become a target of broad interest over the past 15years. With the growing evidence of their important roles in fundamental cellular processes, there is an urgent need to characterize the conformational behavior of IDPs at the highest possible level. The unique feature of NMR spectroscopy in the context of IDPs is its ability to supply details of their structural and temporal alterations at atomic-level resolution. Here, we briefly review recently proposed NMR-based strategies to characterize transient states populated by IDPs and summarize the latest achievements and future prospects in methodological development. Because low chemical shift dispersion represents the major obstacle encountered when studying IDPs by nuclear magnetic resonance, particular attention is paid to techniques allowing one to approach the physical limits of attainable resolution.
PURPOSE: To characterize the relationship between superparamagnetic ferritin-bound iron and diffusion tensor scalars in vitro, and validate the results in vivo. MATERIALS AND METHODS: The in vitro model consisted of a series of 40-mL 1.1% agarose gels doped with ferritin covering and exceeding those concentrations normally found within healthy human gray matter. Additionally, regions of interest were placed in the caudate, putamen, and globus pallidus of 29 healthy volunteer subjects 19-80 years of age. Carr-Purcell-Meiboom-Gill sequence (CPMG) and diffusion tensor imaging (DTI) data were collected at 1.5 Tesla (T) and 3T in vitro, and at 1.5T in vivo. RESULTS: In vitro, linear relationships were observed between ferritin-bound iron concentration, R2 (1/T2 ) and 1/SNR. Eigenvalue repulsion with increasing R2 (decreasing SNR) was reflected in an artifactual increase of fractional anisotropy. In vivo, similar relationships were observed, with mean diffusivity also decreasing linearly with increasing R2 . Lambda 3 showed the strongest correlation with R2 both in vitro and in vivo. CONCLUSION: The observation that DTI metrics correlate with ferritin-bound iron is an important consideration in the design and interpretation of studies exploring the diffusion characteristics of gray matter regions, especially in studies focused on adolescence as well as diseases associated with altered brain-iron load such as pantothenate kinase-associated neurodegeneration, Huntington disease and multiple system atrophy.
- MeSH
- dospělí MeSH
- fantomy radiodiagnostické MeSH
- ferritin metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mozek cytologie metabolismus MeSH
- neurony cytologie metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- senzitivita a specificita MeSH
- techniky in vitro MeSH
- železo metabolismus MeSH
- zobrazování difuzních tenzorů přístrojové vybavení metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mladý dospělý MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
