Solid-State NMR
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Reliable values of the solid-state NMR (SSNMR) parameters together with precise structural data specific for a given amino acid site in an oligopeptide are needed for the proper interpretation of measurements aiming at an understanding of oligopeptides' function. The periodic density functional theory (DFT)-based computations of geometries and SSNMR chemical shielding tensors (CSTs) of solids are shown to be accurate enough to support the SSNMR investigations of suitably chosen models of oriented samples of oligopeptides. This finding is based on a thorough comparison between the DFT and experimental data for a set of tripeptides with both 13Cα and 15Namid CSTs available from the single-crystal SSNMR measurements and covering the three most common secondary structural elements of polypeptides. Thus, the ground is laid for a quantitative description of local spectral parameters of crystalline oligopeptides, as demonstrated for the backbone 15Namid nuclei of samarosporin I, which is a pentadecapeptide (composed of five classical and ten nonproteinogenic amino acids) featuring a strong antimicrobial activity.
- Klíčová slova
- GIPAW, oligopeptides, plane-wave DFT, samarosporin, solid-state NMR,
- MeSH
- aminokyseliny chemie MeSH
- izotopové značení MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- oligopeptidy chemie izolace a purifikace MeSH
- peptidy chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- aminokyseliny MeSH
- oligopeptidy MeSH
- peptidy MeSH
Sensitivity and resolution together determine the quality of NMR spectra in biological solids. For high-resolution structure determination with solid-state NMR, proton-detection emerged as an attractive strategy in the last few years. Recent progress in probe technology has extended the range of available MAS frequencies up to above 100 kHz, enabling the detection of resolved resonances from sidechain protons, which are important reporters of structure. Here we characterise the interplay between MAS frequency in the newly available range of 70-110 kHz and proton content on the spectral quality obtainable on a 1 GHz spectrometer for methyl resonances. Variable degrees of proton densities are tested on microcrystalline samples of the α-spectrin SH3 domain with selectively protonated methyl isotopomers (CH3, CH2D, CHD2) in a perdeuterated matrix. The experimental results are supported by simulations that allow the prediction of the sensitivity outside this experimental frequency window. Our results facilitate the selection of the appropriate labelling scheme at a given MAS rotation frequency.
- Klíčová slova
- CH3 labelling, Magic angle spinning (MAS), Methyl isotopomers, Microcrystalline proteins, Selective deuteration, Solid state NMR,
- MeSH
- deuterium chemie MeSH
- metylace * MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- protony * MeSH
- senzitivita a specificita MeSH
- spektrin chemie MeSH
- src homologní domény MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- deuterium MeSH
- protony * MeSH
- spektrin MeSH
Dipolar recoupling in solid-state NMR is an essential method for establishing correlations between nuclei that are close in space. In applications on protein samples, the traditional experiments like ramped and adiabatic DCP suffer from the fact that dipolar recoupling occurs only within a limited volume of the sample. This selection is dictated by the radiofrequency (rf) field inhomogeneity profile of the excitation solenoidal coil. We employ optimal control strategies to design dipolar recoupling sequences with substantially larger responsive volume and increased sensitivity. We show that it is essential to compensate for additional temporal modulations induced by sample rotation in a spatially inhomogeneous rf field. Such modulations interfere with the pulse sequence and decrease its performance. Using large-scale optimizations we developed pulse schemes for magnetization transfer from amide nitrogen to carbonyl (NCO) as well as aliphatic carbons (NCA). Our experiments yield a signal intensity increased by a factor of 1.5 and 2.0 for NCA and NCO transfers, respectively, compared to conventional ramped DCP sequences. Consistent results were obtained using several biological samples and NMR instruments.
Conducting large-scale solid-state NMR simulations requires fast computer software potentially in combination with efficient computational resources to complete within a reasonable time frame. Such simulations may involve large spin systems, multiple-parameter fitting of experimental spectra, or multiple-pulse experiment design using parameter scan, non-linear optimization, or optimal control procedures. To efficiently accommodate such simulations, we here present an improved version of the widely distributed open-source SIMPSON NMR simulation software package adapted to contemporary high performance hardware setups. The software is optimized for fast performance on standard stand-alone computers, multi-core processors, and large clusters of identical nodes. We describe the novel features for fast computation including internal matrix manipulations, propagator setups and acquisition strategies. For efficient calculation of powder averages, we implemented interpolation method of Alderman, Solum, and Grant, as well as recently introduced fast Wigner transform interpolation technique. The potential of the optimal control toolbox is greatly enhanced by higher precision gradients in combination with the efficient optimization algorithm known as limited memory Broyden-Fletcher-Goldfarb-Shanno. In addition, advanced parallelization can be used in all types of calculations, providing significant time reductions. SIMPSON is thus reflecting current knowledge in the field of numerical simulations of solid-state NMR experiments. The efficiency and novel features are demonstrated on the representative simulations.
Changes in the protonation state of lyophilized proteins can impact structural integrity, chemical stability, and propensity to aggregate upon reconstitution. When a buffer is chosen, the freezing/drying process may result in dramatic changes in the protonation state of the protein due to ionization shift of the buffer. In order to determine whether protonation shifts are occurring, ionizable probes can be added to the formulation. Optical probes (dyes) have shown dramatic ionization changes in lyophilized products, but it is unclear whether the pH indicator is uniform throughout the matrix and whether the change in the pH indicator actually mirrors drug ionization changes. In solid-state NMR (SSNMR) spectroscopy, the chemical shift of the carbonyl carbon in carboxylic acids is very sensitive to the ionization state of the acid. Therefore, SSNMR can be used to measure ionization changes in a lyophilized matrix by employing a small quantity of an isotopically-labeled carboxylic acid species in the formulation. This paper compares the apparent pH of six trehalose-containing lyophilized buffer systems using SSNMR and UV-Vis diffuse reflectance spectroscopy (UVDRS). Both SSNMR and UVDRS results using two different ionization probes (butyric acid and bromocresol purple, respectively) showed little change in apparent acidity compared to the pre-lyophilized solution in a sodium citrate buffer, but a greater change was observed in potassium phosphate, sodium phosphate, and histidine buffers. While the trends between the two methods were similar, there were differences in the numerical values of equivalent pH (pHeq) observed between the two methods. The potential causes contributing to the differences are discussed.
- Klíčová slova
- Diffuse reflectance spectroscopy, Lyophilization, Solid-state NMR spectroscopy, Solid-state acidity, UV/VIS,
- MeSH
- fosfáty * chemie MeSH
- histidin * chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyselina citronová chemie MeSH
- lyofilizace * metody MeSH
- magnetická rezonanční spektroskopie * metody MeSH
- pufry MeSH
- spektrofotometrie ultrafialová metody MeSH
- trehalosa * chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fosfáty * MeSH
- histidin * MeSH
- kyselina citronová MeSH
- pufry MeSH
- trehalosa * MeSH
Understanding the nature of the drug-polymer interactions in micellar drug delivery systems and what happens with the drug and the polymer once the complex has formed is essential for the rational design of the polymeric matrices suitable for a particular drug. In this work, glycopolymeric vesicles-a block copolymer, poly(1-O-methacryloyl-β-d-fructopyranose)-b-poly(methyl methacrylate), (PFru36-PMMA160),-designed to target tumor cells loaded with two drugs, ellipticine and curcumin, were characterized. Advanced solid-state NMR spectroscopy and single-crystal/powder X-ray diffraction (XRD) combined with CASTEP calculations shed light on the nature of the drug, the polymer, and their interactions. While the low drug loading (ca. 5%) ensured that the structure, size, and shape of the polymeric vesicles did not change significantly, the solid-state forms of the drugs changed markedly. Upon loading into the vesicles, ellipticine favored a highly ordered form distinctly different from the bulk drug as indicated by 13C solid-state NMR spectroscopy. A detailed analysis of the CASTEP-calculated 13C spectra derived from crystallographic data based on the lowest mean absolute error showed the best match with form I. Moreover, ellipticine before loading was found as a new polymorph and was described by single-crystal XRD as a new orthorhombic Form III. Likewise, curcumin, originally present in monoclinic Form I was found to recrystallize as metastable orthorhombic Form II inside the vesicles. Intermolecular interactions between the polymeric vesicles and the drugs, ellipticine as well as curcumin, were detected using 2D 1H magic angle spinning experiments, indicating that the drugs are localized in the hydrophobic layer of the vesicles.
- Klíčová slova
- CASTEP calculations, XRD, drug delivery, ellipticine, glycopolymeric vesicles, polymorphism, solid-state NMR,
- MeSH
- glykokonjugáty chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- kurkumin chemie MeSH
- nosiče léků chemie MeSH
- nukleární magnetická rezonance biomolekulární * MeSH
- protinádorové látky chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glykokonjugáty MeSH
- kurkumin MeSH
- nosiče léků MeSH
- protinádorové látky MeSH
We present a detailed analysis of the radiofrequency (RF) field over full volume of a rotor that is generated in a solenoid coil. On top of the usually considered static distribution of amplitudes along the coil axis we describe dynamic radial RF inhomogeneities induced by sample rotation. During magic angle spinning (MAS), the mechanical rotation of the sample about the magic angle, a spin packet travels through areas of different RF fields and experiences periodical modulations of both the RF amplitude and the phase. These modulations become particularly severe at the end regions of the coil where the relative RF amplitude varies up to ±25% and the RF phase changes within ±30°. Using extensive numerical simulations we demonstrate effects of RF inhomogeneity on pulse calibration and for the ramped CP experiment performed at a wide range of MAS rates. In addition, we review various methods to map RF fields using a B0 gradient along the sample (rotor axis) for imaging purposes. Under such a gradient, a nutation experiment provides directly the RF amplitude distribution, a cross polarization experiment images the correlation of the RF fields on the two channels according to the Hartmann-Hahn matching condition, while a spin-lock experiment allows to calibrate the RF amplitude employing the rotary resonance recoupling condition. Knowledge of the RF field distribution in a coil provides key to understand its effects on performance of a pulse sequence at the spectrometer and enables to set robustness requirements in the experimental design.
Solid dispersions of active pharmaceutical ingredients are of increasing interest due to their versatile use. In the present study polyvinylpyrrolidone (PVP), poly[N-(2-hydroxypropyl)-metacrylamide] (pHPMA), poly(2-ethyl-2-oxazoline) (PEOx), and polyethylene glycol (PEG), each in three Mw, were used to demonstrate structural diversity of solid dispersions. Acetylsalicylic acid (ASA) was used as a model drug. Four distinct types of the solid dispersions of ASA were created using a freeze-drying method: (i) crystalline solid dispersions containing nanocrystalline ASA in a crystalline PEG matrix; (ii) amorphous glass suspensions with large ASA crystallites embedded in amorphous pHPMA; (iii) solid solutions with molecularly dispersed ASA in rigid amorphous PVP; and (iv) nanoheterogeneous solid solutions/suspensions containing nanosized ASA clusters dispersed in a semiflexible matrix of PEOx. The obtained structural data confirmed that the type of solid dispersion can be primarily controlled by the chemical constitutions of the applied polymers, while the molecular weight of the polymers had no detectable impact. The molecular structure of the prepared dispersions was characterized using solid-state NMR, wide-angle X-ray scattering (WAXS), and differential scanning calorimetry (DSC). By applying various (1)H-(13)C and (1)H-(1)H correlation experiments combined with T1((1)H) and T1ρ((1)H) relaxation data, the extent of the molecular mixing was determined over a wide range of distances, from intimate intermolecular contacts (0.1-0.5 nm) up to the phase-separated nanodomains reaching ca. 500 nm. Hydrogen-bond interactions between ASA and polymers were probed by the analysis of (13)C and (15)N CP/MAS NMR spectra combined with the measurements of (1)H-(15)N dipolar profiles. Overall potentialities and limitations of individual experimental techniques were thoroughly evaluated.
Recently, proton-detected magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy has become an attractive tool to study the structure and dynamics of insoluble proteins at atomic resolution. The sensitivity of the employed multidimensional experiments can be systematically improved when both transversal components of the magnetization are transferred simultaneously after an evolution period. The method of preservation of equivalent pathways has been explored in solution-state NMR; however, it does not find widespread application due to relaxation issues connected with increased molecular size. We present here for the first time heteronuclear transverse mixing sequences for correlation experiments at moderate and fast MAS frequencies. Optimal control allows to boost the signal-to-noise ratio (SNR) beyond the expected factor of 2 for each indirect dimension. In addition to the carbon-detected sensitivity-enhanced 2D NCA experiment, we present a novel proton-detected, doubly sensitivity-enhanced 3D hCANH pulse sequence for which we observe a 3-fold improvement in SNR compared to the conventional experimental implementation. The sensitivity gain turned out to be essential to unambiguously characterize a minor fibril polymorph of a human lambda-III immunoglobulin light chain protein that escaped detection so far.
- MeSH
- lehké řetězce imunoglobulinů MeSH
- lidé MeSH
- magnetická rezonanční spektroskopie metody MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- proteiny * chemie MeSH
- protony * MeSH
- uhlík MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lehké řetězce imunoglobulinů MeSH
- proteiny * MeSH
- protony * MeSH
- uhlík MeSH
The solid-state NMR measurements play an indispensable role in studies of interactions between biological membranes and peptaibols, which are amphipathic oligopeptides with a high abundance of alpha-aminobutyric acid (Aib). The solid-state NMR investigations are important in establishing the molecular models of the pore forming and antimicrobial properties of peptaibols, but rely on certain simplifications. Some of the underlying assumptions concern the parameters describing the 15N NMR chemical shielding tensor (CST) of the amide nitrogens in Aib and in conventional amino acids. Here the density functional theory (DFT) based calculations were applied to the known crystal structure of one of peptaibols, Ampullosporin A, in order to explicitly describe the variation of the 15N NMR parameters within its backbone. Based on the DFT computational data it was possible to verify the validity of the assumptions previously made about the differences between Aib and other amino acids in the isotropic part of the CST. Also the trends in the magnitudes and orientations of the anisotropic components of the CST, as revealed by the DFT calculations of the full periodic structure of Ampullosporin A, were thoroughly analyzed, and may be employed in future studies of peptaibols.
- MeSH
- antiinfekční látky analýza chemie MeSH
- izotopy dusíku * MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- peptaiboly MeSH
- peptidy analýza chemie MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- ampullosporin MeSH Prohlížeč
- antiinfekční látky MeSH
- izotopy dusíku * MeSH
- Nitrogen-15 MeSH Prohlížeč
- peptaiboly MeSH
- peptidy MeSH
