2D TOCSY and 2D NOESY

1) 2D TOCSY (TOtal Corelation SpectroscopY)

TOtal Correlation SpectroscopY is a method of obtaining relayed connectivities and it is also known by the acronym HOHAHA (Homonuclear Hartmann- Hahn). TOCSY utilizes isotropic mixing to transfer magnetization between spins via the strong scalar coupling, where the resulting magnetization is transferred between all the protons of the coupled spin network. Correlations are seen between distant protons as long as there are coupling between every intervening proton. It is very important to gather information about the side chains of amino acids. It can give information up to 4- 5 bonds (C_α, C_β, C_γ, C_δ) as long as the successive proton is coupled. It is interrupted by zero or small proton-proton coupling or by heteroatoms like oxygen. It requires only single mixing timing and less detailed knowledge of spin system topology.

The magnetisation of the protons via the J_HH happens as given below in the diagram:

The TOCSY experiments can be performed in a numerous ways. The first TOCSY experiment scheme was proposed in 1983 [1]. Then it was improved by changing the mixing schemes [2]. The general pulse sequence diagram for two-dimensional homonuclear magnetization transfer spectroscopy is given here.

Pulse Program from Paper: Pulse sequence for 2D-1H TOCSY

Pulse Program from Paper: Pulse sequence for 2D-1H TOCSY

The gradient-enhanced version of the 2D TOCSY experiment is made by incorporating two pulse field gradients before and after the mixing process. The pulse field gradient for the phase sensitive ge-2D TOCSY with MLEV using echo-antiecho (mlevetgp) and phase-sensitive ge-2D TOCSY with DIPSI-2 using echo-antiecho (dipsi2etgp) is given here.

Pulse Program from Bruker: Pulse program for gradient based phase-sensitive ge-2D TOCSY with MLEV using echo-antiecho (mlevetgp)

Pulse Program from Bruker: Pulse program for gradient based phase-sensitive ge-2D TOCSY with MLEV using echo-antiecho (dipsi2etgp)

2) 2D NOESY (Nuclear Overhauser Effect SpectroscopY)

Generally NMR experiments depend upon coherence transfer via scalar couplings. It will provide coupling between protons of the same amino acid residue. Nuclear Overhauser Spectroscopy is unique among NMR methods. It does not depend on through bond J couplings but depends only on the spatial proximity between protons. When a proton is saturated or inverted spatially, the nearby protons may experience an intensity enhancement. The dipole interactions can be mediated between protons up to a distance of ~5 Å. The sequential assignment process in an unlabelled protein sample is completed using the dipole–dipole cross-relaxation to correlate 1H spins that are close in space. Additionally, distance restraints for structure determination of proteins are derived primarily from 1H–1H dipole–dipole cross-relaxation.

The NOESY experiment trace out through-space proton-proton connectivity:

The pulse sequence for 2D- NOESY experiments is given below. Initially, a 90°–t1–90° element is used to frequency label the spins and return the magnetization to the z-axis. Magnetization transfer occurs via dipolar coupling for a mixing period before observable transverse magnetization is created by the final 90° pulse.The Mixing time, plays a crucial role in these experiments.

Pulse Program from Paper:

The gradient-enhanced version of the 2D NOESY experiment must be recorded in a phase sensitive mode. For this reason, gradients are usually incorporated as a purging method to reduce the needed phase cycle. The delay d8 defines the mixing period in all NOESY experiments. Phase-sensitive 2D NOESY maps can be quickly obtained for concentrated samples in reduced times without unwanted sensitivity losses due to coherence selection and diffusion effects.

Pulse Program from Bruker: The pulse field gradient for the phase sensitive ge-2D NOESY using echo-antiecho (noesyetgp)

Pulse Program from Bruker: Using water flip-back and 3-9-19 (noesyfpgpph19)

The NOE transfer strongly depends on temperature and solvent (viscosity), magnetic field, molecular weight and mixing time. NOE of small molecules are negative and that of large molecules are positive. In small molecules, NOE cross-peaks have opposite phase with respect to diagonal autocorrelation peaks.

The 2D NOESY spectrum of strychnine is given below: 2D-NOESY spectrum of strychnine

References:

1.Braunschweiler, L. and R.R. Ernst, Communication: Coherence transfer byisotropic mixing: Application to proton correlation spectroscopy. Journal of Magnetic Resonance (1969), 1983. 53: p. 521-528.

2.Bax, A. and D.G. Davis, Communication: MLEV-17-based two-dimensionalhomonuclear magnetization transfer spectroscopy. Journal of Magnetic Resonance (1969), 1985. 65: p. 355-360.

3.Griesinger, C., et al., Clean TOCSY for proton spin systemidentification in macromolecules. Journal of the American Chemical Society, 1988. 110(23): p. 7870-7872.

4.Cavanagh, J. and M. Rance, Sensitivity improvement in isotropic mixing(TOCSY) experiments. Journal of Magnetic Resonance (1969), 1990. 88(1): p. 72-85.

5.Cavanagh, J. and M. Rance, Suppression of cross-relaxation effects in TOCSY spectra via a modified DIPSI-2 mixing sequence. Journal of Magnetic Resonance (1969), 1992. 96(3): p. 670-678.

6. Kumar, A., R. Ernst, and K. Wüthrich, Atwo-dimensional nuclear Overhauser enhancement (2D NOE) experiment for theelucidation of complete proton-proton cross-relaxation networks in biologicalmacromolecules. Biochemical and biophysical research communications, 1980. 95(1): p. 1-6.

7.van Zijl, P.C. and R.E. Hurd, Historical Perspective: Gradient enhanced spectroscopy. Journal of Magnetic Resonance, 2011. 213: p. 474-476

8. Neuhaus, D., Nuclear overhauser effect. eMagRes, 2000

9. Bodenhausen, G., et al., Communication: Longitudinal two-spin order in 2D exchange spectroscopy (NOESY). Journal of Magnetic Resonance (1969), 1984. 59: p. 542-550