15N HSQC spectra

Morita, E.H., Shimizu, M., Ogasawara, T. et al. (2004) A novel way of amino acid-specific assignment in1 II-15N HSQC spectra with a wheat germ cell-free protein synthesis system. Journal of Biomolecular NMR, 30 (1) 37 15. 

Fig. 13.2 Expanded region of the overlaid H-15N HSQC spectra from a titration of, 5N-labeled S100B with CapZ peptide. The protein concentration was 1 mM, and the spectra shown are for...

Comparison of1H-15N HSQC Spectra of Yeast Ubiquitin... 

Fig. 2 Chemical shift perturbation and chemical shift mapping, (a) Portions of the [15N, 1H]-HSQC spectra of Bcf-xL recorded in absence (black) and in presence of each of the four molecules (in colors). Resonance assignments for amino acid residues that exhibit large shifts are reported, (b) Structure of Bc1-Xl in complex with the BH3 peptide from Bak (PDB code 1BXL) showing the chemical shift changes in Bcl-xL upon ligand binding (blue, large shits yellow, no shifts the Bak peptide is reported in cyan). Adapted from [48]...

Fig. 9. (a) H, 15N] HSQC NMR spectra of 15N-labeled 7 and 2 mol equiv of 5 -GMP after lmin and 30 min of irradiation (, 195Pt satellites) (b) time dependent decrease in concentration of 7 and formation of 7rG and 7rG2 (lines drawn merely to connect points) (c) quantification of in vitro DNA repair synthesis using an extract prepared from the repair-proficient HeLa cell line. Cisplatin was taken as 100%. Adapted from Ref. (35). 

The solution structures of Raf-, Rif-, and RalGDS-RBD were solved and, in addition, NMR spectroscopy was used to probe the interaction with Ras [211, 213,219]. Only the RBD was labeled with 15N and the shifting of distinct cross peaks (HSQC spectra) due to binding of Ras allowed one to identify the binding surface on the RBD. Consistent with X-ray structures of the complexes (see above) in both Raf and RalGDS, amino acid residues located in (31 and (32 and the C-terminal part of al are involved in the interaction with Ras. 

For the residue-type assignment of the cross peaks, the SH3 domain was expressed with specifically labeled glycine or arginine residues by using amino acid mixtures where only Gly and Arg were 15N-labeled. Again, yields for the labeled proteins were identical to those of the unlabeled product. Analysis of the corresponding [15N,1H]-HSQC spectra... 

Fig. 4.4 Example of amino acid-type selective labeling. A [ H,15N]-HSQC spectrum obtained with the fully 15N-labeled monomeric form of the KSHV protease. B and C [nH,15N]-l-ISQC spectra...

In our experience resonance assignment is speeded up so much in the case of 15N-la-beled peptides that the additional effort in the laboratory is quickly paid for. The HSQC spectra will also help to immediately recognize signal overlap. 

Fig. 8.5 Downfield (a) and upfield (b) compo-nents of 15N doublets of a I PAP-[ H-15N]-HSQC for the protein saposin in Pfl viruses, a results from the subtraction and b from the addition of the spectra containing the in-phase and antiphase components, respectively. The sum of the...

Fig. 14.1 Schematic drawing summarizing fragment screening by NMR. Spectra indicated on the left hand side correspond to [ N HJ-HSQC spectra recorded on 15N-labeled protein in the absence (spectrum 1) or presence (spectrum 2) of a first ligand. Cross peaks in the HSQC that have shifted...

This chapter describes protocols for preparing 15N-labeled proteins (ubiquitin is used as an example) using Escherichia coli cells (with purification) and the wheat germ cell-free system (without purification). A comparison of I I-15N heteronuclear single-quantum coherence (HSQC) spectra of yeast ubiquitin prepared using each method indicates that this wheat germ cell-free system may be used for rapid nuclear magnetic resonance analyses of proteins without purification. 

Figure 7 Heteronuclear correlation spectra (1H-15N HSQC at 500 MHz) of D. melanogaster dUTPase without substrate (A) and with a,p-imino-dUTP. The indicated peaks that change their internal dynamics and diminish, Glyl49-Thrl56 of the C-terminus, are those involved in substrate binding.

Other strategies that show great promise in reducing NMR acquisition time utilise methods to obtain multiple sets of data from one experiment through a concept known as time-shared evolution. An example of this process that should find utility in natural products elucidation was demonstrated by a pulse sequence called CN-HMBC.93 Traditionally, a separate 13C-HMBC and 15N-HMBC were acquired independently. However, the CN-HMBC allows both 13C- and 15N-HMBC spectra to be obtained simultaneously. By acquiring both data sets simultaneously, an effective 50% time reduction can be achieved.93 This approach has also been demonstrated for a sensitivity-enhanced 2D HSQC-TOCSY (heteronuclear multiple bond correlation total correlation spectroscopy) and HSQMBC (heteronuclear single quantum... 

The projection-reconstruction approach is a technique unrelated to covariance processing which can provide data typically inaccessible to the natural product chemist. For example, 13C-15N correlation spectra were obtained for vitamin B12 at natural abundance.104 Compared with a conventional three-dimensional 13C-15N correlation experiment, the projection-reconstruction method provides a sensitivity enhancement of two orders of magnitude. The final 13C-15N spectrum was reconstructed from data obtained from ll l5N and H- C correlations acquired using a time-shared evolution pulse sequence that allowed all the information to be obtained in one experiment.104 Martin and co-workers also demonstrated the ability to generate 13C-15N correlation spectra using unsymmetrical indirect covariance NMR with vinblastine as an example.105 In the latter case, 13C-15N correlation spectra were obtained from - C HSQC data and H-1sN HMBC data that were acquired separately. Both methods provide access to correlations that would be inaccessible for most natural products at natural abundance. 

Figure 2. H hsqC spectra of apoLp-lll. Panel N-uniformly labeled apoLplll. Panel N-backbone labeled apoLp-lll (see text). Panel C,15N-Ieucine specifically labeled apoLp-lll.

As exemplified in Figure 14, the increase in resolution compared to a simple 2D NOESY is dramatic, due in part to the lack of a straightforward correlation between 15N chemical shift and the secondary structure in which a residue is located (in contrast to the case of HN, Ha, and Ca chemical shifts). An analogous combination of TOCSY and HMQC/HSQC yields 3D TOCSY-IIMQC/I ISQC,6 7" where the third dimension as described above shows the chemical shifts of protons to which the amide protons would exhibit correlations in a conventional TOCSY (i.e., those protons in the same spin system). Thus, when satisfactory NOESY-HSQC and TOCSY-HSQC spectra are obtained, a semiclassical route to resonance assignment... 

Figure 14 Comparison of 2D NOESY and 3D 15N-edited NOESY-HSQC spectra of a 41-residue peptide toxin from the Australian funnel-web spider Hadronyche infensa. A strip from the 2D NOESY spectrum is shown on the far left and it illustrates overlapping NOE correlations from three different amide protons (those of Trp13, Lys17, and Gly33). Fortunately, the 15N nuclei for these three amide groups have unique chemical shifts and hence they appear on different 2D planes in the 3D NOESY-HSQC experiment. Strips from these three planes are shown on the right, and they demonstrate that all of the NOE correlations are perfectly resolved in the 3D experiment.

Examples of the application of MCR to both 2D DOSY NMR data23 and 3D [jH-15N] HSQC NMR data24 can be found. In the latter case, the reaction between 15N-labelled cisplatin and the amino acid-nucleotide hybrid (Phac-met-linker p5 dG) is monitored and both analysis of the individual 2D HSQC spectra as well as the simultaneous analysis of all 2D HSQC spectra over time is performed in which case the kinetic reaction profiles can be obtained. The authors found that sub-structures involved in local unfolding as a consequence of the addition of denaturant could be identified, and that this would hardly have been possible without multivariate analysis of the data. 

---