25 research outputs found
Broadband excitation pulses for high-field solid-state nuclear magnetic resonance spectroscopy
In nuclear magnetic resonance spectroscopy, experimental limits due to the radiofrequency transmitter and/or coil means that conventional radiofrequency pulses (“hard pulses”) are sometimes not sufficiently powerful to excite magnetization uniformly over a desired range of frequencies. Effects due to nonuniform excitation are most frequently encountered at high magnetic fields for nuclei with a large range of chemical shifts. Using optimal control theory, we have designed broadband excitation pulses that are suitable for solid-state samples under magic-angle-spinning conditions. These pulses are easy to implement, robust to spinning frequency variations, and radiofrequency inhomogeneities, and only four times as long as a corresponding hard pulse. The utility of these pulses for uniformly exciting 13C nuclei is demonstrated on a 900 MHz (21.1 T) spectrometer
A Comparison of NCO and NCA Transfer Methods for Biological Solid-State NMR Spectroscopy
Three different techniques (adiabatic passage Hartman-Hahn cross-polarization, optimal control designed pulses, and EXPORT) are compared for transferring 15N magnetization to 13C in solid-state NMR experiments under magic-angle-spinning conditions. We demonstrate that, in comparison to adiabatic passage Hartman-Hahn cross-polarization, optimal control transfer pulses achieve similar or better transfer efficiencies for uniformly-13C,15N labeled samples and are generally superior for samples with non-uniform labeling schemes (such as 1,3- and 2-13C glycerol labeling). In addition, the optimal control pulses typically use substantially lower average RF field strengths and are more robust with respect to experimental variation and RF inhomogeneity. Consequently, they are better suited for demanding samples
Ensemble
<p>(<b>top</b>) <b>and cartoon</b> (<b>bottom</b>) <b>representations of S64</b> (<b>left</b>) <b>and S67</b> (<b>right</b>)<b>.</b> The N and C termini are labeled in both the ensemble (top) and cartoon (bottom) representations of the structures of S64 (left) and S67 (right). The three disulfide bonds in the cartoon representations are also labeled. The ensembles represents the 20 structures with the lowest total energy out of 100 calculated structures. The cartoon representations show the lowest energy structure from each ensemble. Note that in the cartoon representation for S64 the third disulfide bond connects to the β sheet (red), not the strand (teal) in front of it.</p
Sequence alignment for the translated sequences of S64 and S67.
<p>This sequence alignment for S64 and S67 illustrates the signal sequence, linker, and mature toxin. Small arrows show the predicted cleavage sites for the mature toxins. The experimentally determined disulfide bond connectivity shown applies to both peptides. Sequences were aligned using ClustalX 2.1 and visualized using JalView 2.7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054401#pone.0054401-Waterhouse1" target="_blank">[36]</a>. The coloring makes use of the default ClustalX color scheme, which is a function of sequence identity and amino acid type.</p
Sequence alignment for mature toxin sequences.
<p>This figure displays the mature toxin sequences for S64 and S67 aligned with representative toxins found using a BLASTp search using the Arachnoserver toxin peptide database. Sequences were aligned using ClustalX 2.1 and visualized using JalView 2.7. The coloring makes use of the default ClustalX color scheme, which is a function of sequence identity and amino acid type.</p
MALDI-TOF mass spectra for unlabeled S64
<p>(<b>left</b>) <b>and S67</b> (<b>right</b>)<b>.</b> The predicted monoisotopic masses for fully oxidized S64 are 3558.3 Da for the [M+H]<sup>+</sup> ion and 1779.69 Da for the [M+2H]<sup>2+</sup> ion. Likewise, the predicted mass for fully oxidized S67 is 2121.41 Da for the [M+2H]<sup>2+</sup> ion. The appearance of peaks at these masses indicates that the cysteines are oxidized for both S64 and S67. The additional peaks seen to the left of the [M+2H]<sup>2+</sup> peak in the S67 mass spectrum correspond to ions that have dehydrated. These peaks are also present in the S64 spectrum but are difficult to discern due to the much wider scale used for this spectrum.</p
<sup>15</sup>N-HMQC spectra for S64
<p>(<b>top</b>) <b>and S67</b> (<b>bottom</b>)<b>.</b> The S64 spectrum includes some minor peaks that are not assigned. These peaks do not have discernable cross-peaks in the NOESY spectra and only very weak cross-peaks in the 3D assignment spectra, which suggest that they are due to unfolded peptide rather than a minor conformation with a different disulfide-bonding pattern.</p
HPLC chromatograms for the cleavage reaction products from S64
<p>(<b>top</b>) <b>and S67</b> (<b>bottom</b>)<b>.</b> The solid line corresponds to the left axis and represents the absorbance at 280 nm; the dashed line corresponds to the right axis and represents the solvent composition. In both chromatograms, the cleaved peptide is well-resolved from other peaks. The large peak that appears at high acetonitrile concentrations corresponds to a combination of the maltose binding domain (MBP) tag, uncleaved fusion protein (MBP:S64/MBP:S67) and TEV protease. Peak identities were confirmed by performing gel electrophoresis and mass spectrometry for select fractions. The flow rate was 4 mL/min (top) and 2 mL/min (bottom).</p