Spectral crowding

In those cases where spectral crowding is very severe, HNCO-based experiments allow the measurement of all or some of the four dipolar couplings (Fig. 8.6) NH, CaC, C N... 

Spectral crowding decreases resolution for larger biomolecules. 

The very high resolution of the chemical shifts gives information on a per-residue basis, and it also leads to spectral crowding in simple experiments. Figure 3 shows three very different ID proton spectra of the 14-kDa protein called a-lactalbumin, in three different states native folded, molten globule, and unfolded (urea denatured). 

In the H- H COSY (correlation spectroscopy) experiment (41), magnetization is transferred via the J-coupling and shows correlations between protons three bonds apart. The cross peak usually shows a characteristic antiphase-square pattern, but it may be split even more by additional passive couplings that lead to spectral crowding and loss of intensity. For small molecules. 

As the size and complexity of a molecule increases, the proton NMR spectrum can easily become crowded and very difficult to interpret. Steroids are a common class of compounds of pharmaceutical interest and provide a very good example of spectral crowding. Steroid NMR spectra typically contain a region... 

Two dimensional experiments can also show correlations between different types of nuclei. These hetero-nuclear experiments have the advantage that nuclei such as and N have much wider chemical shift ranges, and therefore the 2D experiments achieve a tremendous reduction in spectral crowding. The HETCOR (HETeronuclear CORrelation) experiment was the first 2D experiment developed to provide... 

A subjective indication of spectral crowding (at 600 MHz) due to abundance of endogenous metabolites with a wide range of shifts. 

MULTIPLETS in MRS FADE SHAPE SPECTRA with PARTIAL SIGNAL LENGTH Np = 800 (COMPONENTS TOP. TOTAL BOTTOM) SPECTRAL CROWDING and OVERLAPS FREQUENCY BANDS with FOCUS ON DOUBLETS and TRIPLETS... 

The asynchronous contour map was described as helpful for enhancing resolution in the same way 2D correlation spectroscopy does [12] Assuming that the cause of the variations of two coincidentally overlapping peaks are sufficiendy different, asynchronous correlation peaks will be obtained at the corresponding spectral positions, thus spreading the spectral crowding into two dimensions. 

The problem with the application of fliis principle in proteins is spectral crowding for proton interactions and, combined with this, only partial definition of hyperfine lines over the whole range of flie EPR spectrum. This makes direct tensor determination somewhat ambiguous, but in connection with known molecular stractures a unique solution can typically be found. A very important asset in this respect is site-directed mutagenesis, by which specific amino acid residues can be changed. For most of the examples of iron-sulfur clusters discussed, a full analysis was possible even without this tool. 

W.F. Reynolds, R.G. Enriquez, The advantages of forward linear prediction over multiple aliasing for obtaining high-resolution HSQC spectra in systems with extreme spectral crowding, Magn. Reson. Chem. 41 (2003) 927-932. 

In contrast to solution-state NMR spectroscopy, the size of protein assemblies does not affect the line width in solid-state NMR spectroscopy. As only the number of inequivalent amino acids in a sample leads to spectral crowding and resonance overlap, large protein assemblies formed from one or only a few types of monomers are extremely suitable for solid-state NMR spectroscopy. Recent examples include a variety of amyloid fibrils [3, 36, 37], full virus particles [38, 39], and a secretion needle [40]. 

Raman analysis of large macromolecules, such as proteins, often yields complicated, spectrally crowded results. 

Another line of spectroscopic selection options appears if the second spin label is essentially invisible in the pulse EPR measurements. This is the case if chelate complexes of any trivalent paramagnetic lanthanide ions [except Gd(iii)] are used as spin labels. Those lanthanide ions relax fast enough to be non-detectable in any pulse EPR experiment at least down to 10 K. Such a spin label would not lead to any spectral crowding problem, but its dipole-dipole interaction with nitroxide radicals can still be measured via the relaxation enhancement (RE) effect. 

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