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 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

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.

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

NOESY-HSQC

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

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

Triple Resonance Experiments

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

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

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

Structure Generation Using NMR Data from Heteronuclear Experiments