Three-dimensional spectra

Three-dimensional spectra can be conveniently represented by 2D cross-sections taken at appropriate points and perpendicular to one... 

Extractables were further identified by a Hewlett-Packard Model No.5993 GC/MS spectrometry. Solvent extractables were further diluted with Freon TA before GC/MS analysis. Six foot long, 0.3% SP2250 OV17 type material was used as the GC column, with 20-30 cc/min flow rate of helium as the carrier gas. Mass/charge (m/e) units were scanned from 30-800 atomic mass units (amu). Three-dimensional spectra GC/MS were printed out at the end of each run. Results of the GC/MS are shown in Figure 7. 

Figure 13 (a). Chromatogram obtained from injection of pre-treated process liquid from the beginning of the fermentation process, detected at wavelength 245 nm. (b) The three dimensional spectra-chromatogram obtained with the DAD from the same injection as (a). 

The increased resolution that is gained by the addition of another frequency dimension is not without cost. Acquiring the three-dimensional cube is equivalent to acquiring 32 to 128 two-dimensional spectra. If the two-dimensional spectra required 8 h, to obtain, the three-dimensional spectrum will require 24 to 64 h, depending on how many different slices are required and how much time is spent acquiring each slice. However, it is clear that the two-dimensional spectra cannot be analyzed owing to severe overlap of peaks, while the three-dimensional spectra can be analyzed. 

In this case, it is absolutely necessary to label the protein with both C and N. The larger number of residues increase the complexity of N separated three-dimensional spectra such that it is difficult to obtain the assignments by use of dipolar coupling between protons on adjacent residues because of the potentially misleading dipolar coupling to nonadjacent residues. Consequently, it is necessary to employ scalar coupling across the peptide bond to obtain the assigmnents. 

These reduce to the expressions for three-dimensional spectra if P = 0°.) After Fourier transformation a cube that represents the evolution subspace S F F2F ) is created, with the fourth frequency dimension F4 left to the imagination. In this representation the simplest projections are the three first planes F1F4 (where t2 = h = 0), F2F4 (where h = tj, = 0), and F3F4 (where tj = = 0). Resonance... 

Kazimierczuk and co-workers apphed their semi-automatic CLEAN procedure to suppress artifacts in a randomly sampled N-labeled NOESY-HSQC spectrum of ubiquitin [60]. It was demonstrated that the process does not systematically influence relative peak amplitudes, and is therefore applicable to NOESY spectra. Similar conclusions were later drawn by Stanek and Kozmihski [85], and by Werner-AUen and co-workers [84], who compared their reconstructions with conventionally sampled three-dimensional spectra of the same spectral resolution. The algorithm proposed by Kazimierczuk and co-workers was later also applied to higher-dimensional experiments [80]. 

FIG. 9 Background-corrected three-dimensional spectra of flow-injection analysis peaks of horseradish peroxidase (HRP) at = 0.550, 0.715, and 0.850 V versus Ag/AgCl. HRP, 3.2 X 10 M (10 /xL) K2lrCl6 in reservoir, 6.8 x 10 M total flow rate, 0.5 mL min . The inset shows (A) a peak-top total spectrum and (B) a background spectrum at = —0.850 V, where the difference between the two spectra is very small. Even in such a case, reproducible background spectra are obtained. (From Ref. 25.)... 

Robin etal. proposed a processing scheme using ME for the processing of three-dimensional spectra - where ME is applied to the two indirectly detected dimensions prone to truncation, while DFT is used in the last one. They claim to make a gain in acquisition time of a factor of about five. 

The assignment of resonances to the chemical structure commences from the main chain atoms HN, N, CA, CO and CB. The experiments, i.e. the pulse sequences for this purpose, appear superficially complex but are in fact built from concatenated parts of heteronuclear polarization transfers. The magnetization is often derived from the amide proton and also from the amide nitrogen when using transverse relaxation optimized spectroscopy. Subsequently the polarization is relayed to amide nitrogen and further to carbons. Three-dimensional spectra are produced by acquiring the frequencies of the amide proton directly and recording... 

Many such two-dimensional experiments can be performed to correlate various aspects of the behaviour of the spin system. By introducing a second evolution time, three-dimensional spectra can be obtained and so on. 

Three-dimensional spectra allow the spatial structure of complex molecules, e.g., proteins, to be studied (Figure 5). 

Gopinath and Veglia have described a new teehnique for dual aequisition MAS solid-state NMR spectroscopy, which is suitable for simultaneous acquisition of multidimensional spectra of biomacromolecules. Further details of the new method have been presented in another publieation Gopinath and Veglia. It has been shown that the new pulse sehemes enable the simultaneous acquisition of three dimensional spectra of uniformly labelled proteins. The new approach is general and applieable to many 3D experiments, nearly doubling the performance of NMR speetrometers. 

The interpretation of multidimensional spectra can be quite involved, so only a simple overview will be presented here. Two-dimensional spectra are usually represented as contour plots with two frequency axes forming the base plane (e.g., 8h-5h, 5c-5c, or 6c-Jch). and intensity depicted by curves spaced according to the steepness of the ascent these plots resemble geological survey maps. The stacked plot tilts the frequency-frequency plane, showing the peaks as vertical intensity they look like perspective drawings of mountainous terrain. Three-dimensional spectra may be portrayed as false-color (to show intensity) spots in a cube formed by three frequency axes. 

Huang, C. Z. Li, Y. F. Hu, X. L. Li, N. B. Three-dimensional spectra of the long-range assembly of Nile blue sulfate on the molecular surface of DNA and determination of DNA by fight-scattering. Anal. Chim. Acta 1999, 395, 187-197. 

Multidimensional solid-state NMR experiments have been shown to yield completely resolved spectra of uniformly labelled proteins in oriented lipid bilayers. In three-dimensional spectra, each amide resonance is characterized by three frequencies ( H chemical shift, chemical shift and H-i N heteronuclear dipolar coupling), which provide the source of resolution among the various sites as well as the basic input for structure determination based on orientational constraints. The data shown in Figure 5 are from a 50-residue protein in oriented lipid bilayers. More importantly, since the polypeptides are immobilized by the lipids on the relevant NMR time-scales, there can be no further degradation of line widths or other spectroscopic properties as the size of the polypeptide increases. Although larger proteins will have more complex spectra resulting from the increased number of resonances, there is no fundamental size limitation to solid-state NMR studies of membrane proteins. 

---