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Chapter 16. 3D Experiments

Chapter 16. 3D Experiments. 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

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Chapter 16. 3D Experiments

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  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 andcarbon sources.

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

  5. NOESY-HSQC

  6. 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

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

  8. Triple Resonance Experiments

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

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

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

  12. Structure Generation Using NMR Data from Heteronuclear Experiments

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