HNCA assignment experiments

Although, the MP-HNCA-TROSY experiment alone can yield sequential assignment, it can be also used concomitantly with the HNCA-TROSY experiment. This strategy is explained later, but let us first focus on the coherence transfer efficiency of the MP-HNCA-TROSY experiment. The transfer functions for the antiphase experiment (the efficiency for the in-phase experiment is practically the same) are calculated according to Eqs. (10) and (11) for the intraresidual... 

Fig. 21.4 Three-dimensional structure of the enzyme cytochrome P450 oxidoreductase. The cofactors FAD and FMN are depicted in light blue. Loop regions are represented by cylindrical rods (yellow) a-helices and /(-sheets (in white) are represented by ribbons and arrows, respectively. Resonance assignments for the residues located in the key loops regions [30] highlighted in purple and red were obtained with a 3D SEA-HNCA-TROSY experiment.

NMR spectroscopy of biological macromolecules, Inmr(-Pi. fb. 3) data from 3D correlation experiments is normally sufficient to achieve this. For instance, the output of the 3D HNCA correlation experiment should ensure that every HN nucleus in a protein under investigation has a unique and unambiguous resonance assignment dehned in the first instance by its chemical shift but also by the chemical shifts of spin-coupled/correlated and nuclei. In effect, each HN nucleus is referenced by a completely unique 3D address, HN(Fi5n, Ri3c Rih). defined by three resonance frequencies. 

The larger the protein, the greater the resonance overlap. Often several different residues have degenerate 13Ca frequencies which will make an unambiguous assignment difficult or even impossible. In these cases, additional information from different NMR experiments is required. One possibility is to use the carbonyl chemical shift instead of the Ca chemical shift and measure the HNCO/HN(CA)CO pulse sequence pair [37, 45, 46, 49, 50]. As with the HNCA/HN(CO)CA combination, one of the experiments, the... 

Fig. 10.5 Sequential resonance assignment of the polypeptide backbone of 2H, 3C, 5N-labeled DHNA using the HNCA triple resonance experiment, which connects the Hn and 15N resonances of the amide groups with the sequential and intraresidual 13C chemical shifts. The dotted...

Figure 15 Schematic illustration of three different pairs of triple resonance NMR experiments that can be used for making sequence-specific resonance assignments. Left panel HNCACO and HNCO middle panel HNCA and HN(CO)CA right panel HNCACB and CBCA(CO)NH. In each case, the experiment listed first, which is shown in red, provides intraresidue correlations (and sometimes also interresidue correlations), whereas the experiment listed second, shown in blue, provides only interresidue correlations.

The workings of the 3D HNCA experiment are illustrated diagrammatically in Figure 5.24. Frequently, HNCA spectral data is displayed in the form of individual C-, frequency (F2 and F3) contour maps resolved at different frequencies (H) (all in ppm). However, data could equally well be plotted in the form of H-frequency (Fi and F3) contour maps if this were more helpful for assignment purposes. Usually the HNCA experiment is combined with other 3D correlation experiments with names such as HNCO, HCACO and... 

HCA(CO)N experiments. These names are deliberately descriptive and should suggest the correlations that these experiments are intended to establish by analogy with the HNCA experiment described here. All these correlation experiments acting in concert are designed to ensure optimal and mostly complete, unique and unambiguous assignments of Tf-resonance peaks to nuclei in given protein NMR spectra. This level of information is usually sufficient... 

Proton-detected scalar coupling based assignment strategies in solid-state MAS NMR spectroscopy applied to perdeuterated proteins have been presented by Linser et al Assignment of proteins in solid-state NMR relies mainly on correlations among heteronuclei. This strategy is based on well dispersed resonances in the dimension. In many eomplex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. Linser et al. have shown that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. 

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