HNCO experiments

A suitable CCR-rate to determine the backbone torsion angle 0 by CCR is the /NHCHcf dipole-dipole CCR-rate that conveniently can be measured by an HNCA-derived experiment [44]. Alternatively, like for the torsion angle 0, the FcuaCii-i) dipole-CSA CCR can be measured by a triple-resonance experiment that is derived from a combination of HNCA and HNCO experiments [45]. Also, CCR experiments for which the rate depends on 0 and

dipole-dipole CCR experiment can be used [46]. Unfortunately for the peptide under investigation, we were not able to successfully record any of these spectra, possibly due to the relatively strong auto relaxation. 

To illustrate how a complex triple-resonance pulse sequence can be understood relatively easily using the concepts developed in this book, consider the HNCO experiment. The Hn and 15N chemical shifts of one residue are correlated with the chemical shift of the carbonyl group of the previous residue (Fig. 12.56). This is accomplished by a simple out-and-back scheme of coherence transfer (Fig. 12.57) xHn 15N 13C (H) 15N fe) xHn... 

Chen K, Tjandra N (2009) Direct measurements of protein backbone 15 N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment. J Magn Reson 197 71-76... 

HNCO experiment, modified to include CPMG relaxation period. The effect from couplings was minimised by using band-selective pulses in the CPMG pulse-train. The dispersion curves of CO are sensitive to the hydrogen bond dynamics. 

The reduced dimensionahty approach turned out to be beneficial for coupling constants measurements. Kozminski et al. described a C -coupled-MQ-HNCO experiment for a robust evaluation of J(Ni,C i i ) couplings that can be used for the secondary structure identification. The sequence includes nested HMQC coherence transfer blocks generating multiple-quantum coherence. The evolution of the coherence is recorded... 

Fig. 4. The HNCO-TROSY experiment for recording solely interresidual 1HN, 15N, 13C correlations in 13C/15N/2H labelled proteins. All 90° (180°) pulses for the 13C and 13C spins are applied with a strength of 2/ /l5 (p/ /3), where 2 is the frequency difference between the centres of the 13C and 13Ca regions. All 13Ca pulses are applied off-resonance with phase modulation by Q. A = 1/(4/hn) Tn = l/(4/NC ) S = gradient + field recovery delay 0 < k < TN/z2,max- Phase cycling i = y 4>2 = x, — x + States-TPPI 03 = x 0rec = x, — x.

In the alternative approach, the HN(i), 15N( j, 13C (i/i— 1) correlations in the HNCA-TROSY spectrum can be supplemented with the data from the HN(CO)CA-TROSY experiment72 73 yielding solely 11 IN(/), 15N( ), 13C (i- 1) correlations. To this end, the HNCO-TROSY experiment is extended with the 13C —> 13C INEPT step, which utilizes rather large (ca. 51-55 Hz) one-bond scalar coupling between the 13C and 13C spins in order to transfer magnetization from the 13C (< — 1) nucleus further to the 13C ( — 1) spin. 

The HNCA-TROSY experiment can be readily extended to correlate the 13C spin of the preceding residue with intraresidual 11 IN(/), 15N(i), and 13C° (/ ) frequencies. This kind of four-dimensional HNCO CA-TROSY experiment (Fig. 7) was recently introduced by Konrat et al.79 The coherence flows through the following pathway... 

HNfz) connectivities with good sensitivity, excluding serines, threonines, leucines, and glycines (and probably valines and prolines). Thus, the use of HNCO-TROSY/HN(CA)CO-TROSY experiment pair can be very useful in assigning high molecular weight proteins. 

The set of four-dimensional (HNCO, iCA, HNCOCA and HNCACO) TROSY experiments has been successfully applied in the assignment of several large proteins.12,25,26 These three experiments are able to resolve... 

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... 

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... 

Fig. 8.6 Schematic representation of the modified experiments HNCO (a) and (HA)CA(CO)NH (b) to measure residual dipolar couplings. The white bars represent the extra pulses that are applied in an interleaved manner to collect the in-phase 15N magnetization. The anti-phase 15N...

During the first month of this experiment, it was realized that this reaction is extremely variable. Thus, diverse amines (ammonia, primary and secondary amines, hydrazine derivatives, hydroxylamines) 13, carbonyl compounds (aldehydes, ketones) 14, acid components 15 or their anions (H2O, Na2S203, H2Se, R2NH, RHN-CN, HN3, HNCO, HNCS, RCO2H, RCOSH, ROCO2H, etc.), and the isocyanides could form the a-adducts 16 that rearrange into their products 17 (Scheme 1.5). 

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