Two‐, Three‐ and Four‐ Dimensional Nuclear Magnetic Resonance of Biomolecules

Abstract

AbstractNuclear magnetic resonance (NMR) is associated with transitions, induced by radiofrequency (RF) irradiation, between energy levels of nuclei with nonzero spin quantum numbers in a magnetic field. Traditional “one‐dimensional” (1D) NMR spectra are presented as a plot of signal intensity versus applied frequency and provide information about the chemical environment of magnetically active nuclei. This is used to deduce information about the chemical structure and dynamics of molecules containing the magnetically active nuclei (most often protons). The frequencies of individual nuclei are referred to as their chemical shifts. Multidimensional NMR encompasses a range of related techniques, based on application of a variety of precisely timed RF pulses to the sample, which extends traditional 1D NMR into two, three, or four frequency dimensions. These additional frequency dimensions may be the same as the first frequency dimension, referred to as homonuclear multidimensional NMR, or different, referred to as heteronuclear NMR. In multidimensional NMR of biomolecules the most common frequencies correspond to1H in the directly detected dimension and one or more of1H,13C or15N in the additional dimensions. Peaks in such spectra are defined by their intensity and by their frequency coordinates in two, three, or four dimensions obtained by extrapolation back to the respective frequency axes, which are in turn determined by the nature of the applied RF pulse sequences. The intensity and chemical shifts of such peaks provide information about chemical environment, molecular connectivity, or spatial proximity of the participating nuclei. Multidimensional NMR has major advantages over 1D NMR: reduction of peak overlap and provision of information on connectivity (either through bonds or through space). Multidimensional NMR of biomolecules is usually done for samples in solution at millimolar concentration. Where experiments require the use of13C or15N frequencies, it is usually necessary to enrich isotopically the macromolecule with these nuclei to >90%. This is readily achieved with modern molecular biology techniques and does not present a major limitation. The biggest limitation relates to molecular size, with studies generally limited to macromolecules of less than approximately 35 kDa.