1. Chapter 16. 3D Experiments

2. Heteronuclear methods are needed in the study of 'larger' proteins
For proteins less than about 100 residues, conventional homonuclear
2D NMR methods (DQFCOSY, NOESY, etc.) can be applied for
structure determination purposes. As the number of residues and
molecular mass increases beyond 100 and 12 kDa, respectively, two
main obstacles present themselves which prevent the extension of
2D NMR methods to these systems:
Increased spectral complexity arising from the larger number of
protons results in extensive resonance overlap and degeneracy,
rendering 2D homonuclear spectra uninterpretable.
(2) The rotational correlation time, τc, increases with molecular
weight, resulting in large 1H line widths and a concomitant decrease
in the sensitivity of correlation

3. These obstacles can be overcome by increasing the dimensionality of the spectra toresolve problems of spectral overlap, and by simultaneously making use of heteronuclear couplings that are larger than the line widths to circumvent limitations in sensitivity. This necessitates the use of uniformly 15N and/or 13C-labeled proteins, which may be produced by expressing cloned proteins in bacterial systems on minimal media supplemented by 15NH4Cl and/or 13C-glucose as the sole nitrogen and carbon sources.

4. Summary of one-bond heteronuclear couplings along the polypeptide chain utilized in 3D and 4D NMR experiments

5. NOESY-HSQC

7. A schematic illustration showing the relation between a 3D 1H-15N NOESY-HSQC
experiment and 2D 1H-1H NOESY (F1, F3) and 2D 1H-15N HSQC (F2,F3) spectrum

8. F2 (15N) planes from a 3D 1H-15N NOESY-HSQC experiment

9. Triple Resonance Experiments

21. Pulse sequence for the HN(CO)CA triple resonance experiment

23. F1 (15N) planes from H(CA)NH, HN(CO)CA and HNCA triple resonance experiments

24. F2 (15N) plane from a CBCA(CO)NH triple resonance experiment

30. Structure Generation Using NMR Data from Heteronuclear Experiments