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Multidimensional correlation experiments can be arduous to analyse at the best of times Therefore, any practitioner of biomolecular NMR spectroscopy will want to do the minimum number of experiments to achieve unique and unambiguous resonance assignment of as many amino-acid residue 1 H nuclei as necessary in order to enable the critical NOESY experiments Two of the simplest 3D correlation experiments that have been used are 3D 1 H 15 N TOCSYHSQC and 3D HCCH COSY/TOCSY Such 3D correlation experiments are known as doubleresonance experiments in that they generate intensity data I(F1 , F2 , F3 ) emanating from the double resonance of two entirely different populations of nuclei, either 1 H and 15 N nuclei, or 1 H and 13 C nuclei respectively The workings of the 3D 1 H 15 N TOCSY-HSQC experiment are illustrated diagrammatically in Figure 520.

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The pulse sequence is designed to develop transverse magnetisation in aliphatic 1 H nuclei (speci cally C -1 H atoms) (source nuclei I) (F1 ) that evolves according to chemical shift Spin spin coupling then enables coherence transfer involving both intraand inter-amino-acid residue 1 H15 N nuclei Heteronuclear multiple and single quantum coherence (HMQC/HSQC) is generated involving 15 N nuclei (correlating heteronuclei) (F2 ) that is subsequently converted back into transverse magnetisation involving 1 H15 N nuclei 1 15 H N magnetisation becomes modulated by the chemical shift of the directly attached 15 N nuclei and then nal coherence is observed involving 1 H15 N nuclei (destination nuclei S) (F3 ) Spectral data is usually displayed in the form of individual 1 H 1 H frequency (F1 and F3 ) contour maps resolved at different 15 N frequencies (F2 ) (all in ppm) (Figure 521).

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Vertically correlated cross-peaks allow for the assignment of neighbouring amino-acid residue C -1 H (i, i 1) and 1 H15 N (i) resonance signals, as a function of 15 N chemical shift By comparison, 3D HCCH COSY experiments are a little simpler (Figure 522) An initial pulse develops transverse magnetisation in aliphatic 1 H nuclei (speci cally 13 C -1 H atoms) (source nuclei I) (F1 ) that evolves according to chemical shift before polarisation transfer.

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