Buildup rates of the nuclear Overhauser effect measured by two-dimensional proton magnetic resonance spectroscopy: implications for studies of protein conformation

Abstract

It is demonstrated, by means of experiments with the basic pancreatic trypsin inhibitor, that the buildup rates of the nuclear Overhauser effect can be measured by two-dimensional NMR spectroscopy. Qualitative correlations between the buildup rates of first-order Overhauser effects, which arise from direct dipole-dipole coupling between closely spaced protons, and the proton-proton distances in the protein conformation are established. Second-order Overhauser effects due to spin diffusion by cross-relaxation between more distant protons are also identified. On the basis of these observations, potentialities and limitations of two-dimensional nuclear Overhauser enhancement spectroscopy for studies of the conformation of biological macromolecules are discussed and suggestions made for improved experimental procedures. For quantitative measurements of Overhauser effects, the use of phase-sensitive spectra and of techniques for selective suppression of J cross-peaks in data sets recorded with very short mixing times appears particularly important.