2D HSQC

Nolls, P., Parella, T. Spin-edited 2D HSQC-TOCSY experiments for the measurement of homonuclear and heteronuclear coupling constants application to carbohydrates and peptides. /. Magn. Reson. 2005, 176, 15-26. 

Backbone dynamics are most commonly investigated by measurement of 15N T and T% relaxation times and the fyH -15N NOE in uniformly 15N-labeled protein. To circumvent problems associated with the limited dispersion of the NMR spectra of unfolded proteins, the relaxation and NOE data are generally measured using 2D HSQC-based methods (Farrow et al., 1994 Palmer et al., 1991). 

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... 

The 50 highest scoring compounds in the ESI-MS analyses were each evaluated by 2D HSQC NMR in the presence of RGS4 protein (see Section... 

The coupling of the GPC spin column/ESI-MS screening results with NMR (2D HSQC) is a powerful method for confirming that the non-covalent binders identified by the MS experiments truly bind at the predicted active site by observing NMR chemical shift perturbations in the vicinity of the protein active site [1, 15]. In contrast, the absence of chemical shift perturbations or a random distribution of chemical shift changes on the protein surface would imply a lack of an interaction of the compound with the protein or potentially the existence of non-specific binding. The development of the GPC spin column/MS/NMR assay... 

The two compounds with MWs 145 Da and 155 Da identified by GPC spin column/ESI-MS as non-covalent binders to MMP-1 were then each analyzed in the presence of MMP-1 by 2D HSQC NMR (Fig. 2.24). Chemical shift... 

GENERAL APPEARANCE OF INVERSE 2D SPECTRA 11.2.1 2D HETCOR versus 2D HSQC/HMQC... 

N = 113.2 ppm in the 2D HSQC (Fig. 12.51). In practice, the 3D matrix is projected onto the floor of the 3D cube (Fig. 12.49) and the point values (column and tier numbers) are read off of this low-resolution 2D HSQC spectrum for each crosspeak T15 is centered at point numbers 189 (column = F3 = Hn) and 74 (tier = Fi = 15N). From this position on the floor, the spin system rises up in a vertical line along the / j (rows) dimension. 

Strip plots can only be constructed when the crosspeaks have already been assigned in the 2D HSQC spectrum. In a 15N-labeled protein, sequence-specific assignments come from sequential NOE (a,N, /3,N and N,N) crosspeaks located in the 3D HSQC-NOESY spectrum. The walk through the protein backbone is done in the same way as with unlabeled proteins, except that overlap in NOESY spectra is greatly reduced by spreading the crosspeaks out in the 15N dimension of a 3D spectrum. 

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