HSQC spectrum

The method is more sensitive due to the higher proton multiplicity and is suitable for screening proteins up to 40kDa. The key binding site residues of a protein can be identified by using a known inhibitor to identify cross-peaks in the HSQC spectrum [38]. 

Fig. 2. (Left) 750-MHz 15N- H HSQC spectrum and (right) 750-MHz HNCO spectrum of apomyoglobin at pH 2.3,10 mM acetate-

Interpreting HMQC/HSQC spectra is relatively straightforward as you can see from the HSQC spectrum of the morpholine compound (Spectrum 9.3). Basically, it s a case of lining up the proton signal with the contour, and reading off the 13C chemical shift. The technique is extremely powerful - particularly when used in combination with HMBC as we ll see later. In examples like this one, it... 

Using strychnine (1) as a model compound, a pair of HSQC spectra are shown in Fig. 10.16. The top panel shows the HSQC spectrum of strychnine without multiplicity editing. All resonances have positive phase. The pulse sequence used is that shown in Fig. 10.15 with the pulse sequence operator enclosed in the box eliminated. In contrast, the multiplicity-edited variant of the experiment is shown in the bottom panel. The pulse sequence operator is comprised of a pair of 180° pulses simultaneously applied to both H and 13C. These pulses are flanked by the delays, A = l/2(xJcii), which invert the magnetization for the methylene signals (red contours in Fig. 10.16B), while leaving methine and methyl resonances (positive phase, black contours) unaffected. Other less commonly used direct heteronuclear shift correlation experiments have been described in the literature [47]. 

Fig. 10.16. (A) GHSQC spectrum of strychnine (1) using the pulse sequence shown in Fig. 10.15 without multiplicity editing. (B) Multiplicity-edited GHSQC spectrum of strychinine showing methylene resonances (red contours) inverted with methine resonances (black contours) with positive phase. (Strychnine has no methyl resonances.) Multiplicity-editing does have some cost in sensitivity, estimated to be 20% by the authors. For this reason, when severely sample limited, it is preferable to record an HSQC spectrum without multiplicity editing. Likewise, there is a sensitivity cost associated with the use of gradient based pulse sequences. For extremely small quantities of sample, non-gradient experiments are preferable.

Fig. 1.9 [ 5 N. HJ-HSQC spectrum of 15N-uniformly labeled SH3 domain (A) and of a sample selectively...

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...

Fig. 5.7 Ratio of intensities of cross peaks in the spin label (a). Ratio of intensities in the presence [15N, H]-HSQC spectrum in the presence of5-dox- of both spin label and 5 mM CaCI2 to those in the...

Fig. 19.4 a Plot of the amino acid sequence versus differences in chemical shift of the H- N cross peaks in the 2D HSQC spectrum of the 24 kD fragment of GyrB (23-234) upon complexation with the compound Ro 05-8733. b X-ray structure of the complex of the GyrB fragment and... 

Fig. 2 Comparison of an HSQC spectrum of a purified in vitro sample of NmerA (left) with an in-cell spectrum of NmerA (right). The level of background signals in the in-cell spectrum is low, allowing the observation of the protein in its natural environment...

The order in which various NMR data are acquired is largely one of user preference. Acquisition of the proton reference spectrum will invariably be undertaken first. Whether a user next seeks to establish homo- or heteronuclear shift correlations is where individual preferences come into play. Many spectro-scopists proceed from the proton reference spectrum to either a COSY or a TOCS Y spectrum next, while others may prefer to establish direct proton-carbon chemical shift correlations. This author s preference is for the latter approach. From a multiplicity-edited HSQC spectrum you obtain not only the carbon chemical shifts, which give an indication of the location of heteroatoms, the degree of unsaturation and the like, but also the number of directly attached protons, which eliminates the need for the acquisition of a DEPT spectrum [51, 52]. The statement in the prior sentence presupposes, of course, that there the sensitivity losses associated with the acquisition of multiplicity-edited HSQC data are tolerable. 

Fig. 2. N HSQC spectrum of a 75 mM solution of Pro -cyclosporin in CDCI3 at natural isotope abundance using the pulse sequence of fig. 1 without N decoupling during acquisition, t = 5.7 ms, SL = 2.5 ms. An additional, short spin-lock pulse was used right before signal detection [8]. The projections are shown at the top and on the left. (Reproduced by permission of Academic Press from...

Figure 4 Natural Abundance H— 5N HSQC Spectrum (600 MHz, 300K, DMSO-d6, Total Measuring Time = 2 h) of the Cyclic Pentapeptide Sequence c[-Asn-Glu-D-His-Ala-Gly-]a...

Load the spectrum of the gradient-assisted, inverse detected, 2D CH-HSQC-TOCSY experiment acquired with the echo-antiecho technique, D NMRDATA GLUCOSE 2D CH GCHICOTO 001999.RR. Check and if necessary correct its calibration in both dimensions. Set up a layout as for the basic HSQC spectrum. Compare the spectrum with the spectra of the basic HSQC and HMQC experiments. Use the same rows or columns to identify the additional TOCSY-peaks. 

Figure 3-28 A15N - H HSQC spectrum of partially denatured 129-residue hen lysozyme. Boxes enclose the tryptophan indole region (upper left), the arginine side chain NE region (upper left), and a portion of the amide NH region (lower center and enlarged in the insert). Resonances of pairs of hydrogen atoms in side chain (Asn and Gin) amide groups are indicated by horizontal lines. From Buck et al.52i...

Record the 2-D H-13C heteronuclear single quantum coherence (HSQC) spectrum (Braun et al., 1998, pp. 497-500). 

Fig. 9.2 (a) H spectrum from C70H38. (b) The corresponding ICC HSQC spectrum... 

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