NOESY-HMQC

Sequential assignments TOCSY-NOESY NOESY-HMQC TOCSY-NOESY HCACO HNCA HNCO HCA(CO)N... 

Identi6cation NOESY-NOESY NOESY-HMQC 3D NOESY-HMQC... 

Ikura M, Kay LE, Tsudin R, Bax A. Three-dimensional NOESY-HMQC spectroscopy of a 13C-labelled protein. J Magn Reson 1990 86 204-209. 

D 15N-edited NOESY (15N-NOESY-HMQC) provides imino-imino, imino-amino and aromatic-imino/amino connectivities. The approach often fails in regions such as loops and bulges where bases do not form hydrogen-bonded pairs. In that case, imino assignment can be obtained from... 

D 13C-edited NOESY (13C-NOESY-HMQC) identifies sugar-to-base connectivity in a manner similar to homonuclear NOESY spectra. The intra/inter-residue ambiguity can be resolved with the help of... 

HSQC-ROESY, HMQC-NOESY, HMQC-ROESY.49... 

S.6 Two- and Three-Dimensional HSQC-TOCSY, HSQC-NOESY, HSQC-ROESY, HMQC-NOESY, HMQC-ROESY... 

Figure 12.12a gives a good illustration of the need for going to a third dimension to facilitate the interpretation of a crowded 2D spectrum. The NOESY spectrum of a uniformly 15N-enriched protein, staphylococcal nuclease, has so many cross peaks that interpretation is virtually impossible. However, it is possible to use, 5N chemical shifts to edit this spectrum, as indicated in Fig. 12.121) and c in a three-dimensional experiment. With the 15N enrichment, NOESY can be combined with a heteronuclear correlation experiment, in this case HMQC, but HSQC could also be used. A 3D pulse sequence can be obtained from two separate 2D experiments by deleting the detection period of one experiment and the preparation period of the other to obtain two evolution periods (q and t2) and one detection period (f3). In principle, the two 2D components can be placed in either order. For the NOESY-HMQC experiment, either order works well, but in some instances coherence transfer proceeds more efficiendy with a particular arrangement of the component experiments. We look first at the NOESY-HMQC sequence, for which a pulse sequence is given in Fig. 12.13. The three types of spins are designated I and S (as usual), both of which are H in the current example, and T, which is 15N in this case.

FIGURE 12.12 (a) NOESY spectrum (500 MHz) of staphylococcal nuclease, uniformly enriched in 15N. (b) Two adjacent planes from a NOESY-HMQC experiment with staphylococcal nuclease, separated by 15N chemical shift difference of 0.9 ppm. Adapted from Marion et a/.118... 

Application of NOESY-HMQC to the nuclease sample replaces the 2D NOESY spectrum of Fig. 12.12a by a 3D spectrum that can be displayed in a cube but is more easily interpreted as a set of planes, as indicated schematically in Fig. 12.14. Two of the NOESY planes obtained in the nuclease experiment are illustrated in Fig. 12.126. Clearly, the 3D experiment is successful in editing the uninterpretable spectrum of Fig. 12.12a into manageable pieces. Moreover, each NOESY plane is labeled by the chemical shift of the 15N that is coupled to one of the protons, so additional useful information may be available if that 15N chemical shift can be related to structural features in the molecule. Note that a proton not coupled to any 15N generates an axial peak, rather than a cross peak, in the 3D spectrum, but phase cycling is used to remove axial peaks. Also, displays other... 

FIGURE 12.13 Pulse sequence for the three-dimensional experiment NOESY-HMQC. The first three 90° H pulses constitute the usual NOESY sequence, with mixing time r.The 180° 15N pulse at time 3 removes the effect of H-15N couplings. The H and 15N pulses at times 7, 9, 11, and 13 constitute the HMQC sequence, with the pulse at time 7 serving as part of both sequences. 

Write a coherence pathway for the four-dimensional HMQC-NOESY-HMQC experiment, based on the pulse sequence in Fig. 12.15. 

Stractnral conhrmation and complete NMR assignments were accomplished by 3D PFG NOESY-HMQC experiments nsing a C-emiched sample (Satake et al. 1995). Althongh the 3D NMR techniqne has become a rontine method for studies of protein and nucleotide, apphcations to natural products are rate. Unlike proteins, it is very difficult to enrich marine natural products with more than 90% abnndance. Moreover, the stractural elucidation of most natural products can be accomplished by 2D NMR experiments. MTX needed 3D NMR experiments becanse more than 200 proton signals give rise to over 2000 cross peaks in the 2D NOESY spectra. 

Figure 6-36 The pulse sequence for the three-dimensional NOESY/HMQC experiment.

This type of heteronuclear 3D experiment is called NOESY-HMQC. (In Figure 6-37, there are two H dimensions and one N dimension.) Most 3D experiments use high-sensitivity methods and displays that are particularly effective for large molecules. Thus, COSY is not often used, but TOCSY-HMQC is a useful method for separating H- H coupling connectivities into a C or " N dimension. The homonuclear 3D experiment NOESY-TOCSY (all three dimensions are H) separates through-space connectivities from... 

Figure 10 Portions of NOESY spectra and 1D slices through the frequencies of aromatic protons, (a) A 150-ms 2D NOESY spectrum of a 27-nt DNA stem-loop 93 a slice through the frequency of A5H8 is shown, (b) A 200-ms 2D NOESY spectrum of a 34-nt RNA stem-loop 68 a slice through the frequency of C7H6 is shown. Assignments of H5 and H5" protons are tentative. Note that some of the cross-peaks partially overlap with cross-peaks in another slice through the frequency of A8H2. (c) A 150-ms 3D 13C-edited NOESY-HMQC spectrum of the same molecule shown in (b). A slice of the proton and carbon frequencies of H6 and C6 in residue C7 are shown. Note a significantly lower digital resolution in the indirect uj2 dimension in this spectrum compared to the indirect u1 dimension in the 2D NOESY spectrum shown in (b).

Concatenation of the 3H—15N HSQC (or HMQC) sequence with a JH—JH NOESY gives rise to the 3D 15N-edited NOESY-HSQC (or 3D NOESY-HMQC) experiment.66-68 Here, two of the frequency dimensions represent the amide JH and 15N chemical shifts, while the third dimension provides information about the chemical shift of protons with which each amide proton is dipolar coupled (i.e., separated by <5.5 A). The spectrum is routinely viewed as narrow 2D (JH—JH) strips taken at the 15N chemical shift of each crosspeak in the JH—15N HSQC spectrum (see Figure 14). 

Bernstein, R., et al., Computer-Assisted Assignment of Multidimensional NMR Spectra of Proteins Application to 3D NOESY-HMQC and TOCSY-HMQC Spectra, J. Biomol. NMR, 3, 245, 1993. 

The 3D HMQC-NOESY-HMQC (HSQC) experiment together with 4D -edited... 

NOESY-HMQC (HSQC) experiment (b) 4D i C/i W-edited and i C/ C-edited NOESY experiments. Colour notation is identical with Fig. 5.17. 

Figure 5.27 Two 2D contour maps [/NMaC i, 2)] obtained by a HMQC-NOESY-HMQC...

NOESY-HMQC (HSQC) double resonance experiments can provide unique and unambiguous indications of inter-residue proximities by through-space NOE correlations between pairs of IV H resonance signals. The appearance of symmetrical peaks (see triangulations above) occurs when NOEs are observed between NH systems of amino acid residues with essentially identical chemical shifts to each other (adapted from J. Am. Chem. Soc., 1990, 112, 9020-9022 fig. 2). 

Satake, M., S. Ishida, and T.J. Yasumoto, Structural confirmation of maitotoxin based on complete 13C NMR assignments and the three-dimensional PFG NOESY-HMQC spectrum. JAm Chem Soc, 1975 117 7019. 

Melting points were taking on Yamazawa micro-melting point apparatus optical rotations were measured on a JASCO-360 digital polarimeter UV spectra were obtained on a Hitachi 200-10 spectrophotometer IR spectra were taken on a JASCO IR-A-2 spectrometer HNMR, 13CNMR, IH-H COSY, NOESY, HMQC and HMBC spectra were taken on a Bruker AM-400, Bruker AM-500 MS were obtained on Hitachi Rmu -7M spectrometer. 

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