Three-dimensional NMR spectroscopy of a protein in solution

Abstract

The geometric information used to solve three-dimensional (3D) structures of proteins by NMR spectroscopy resides in short (<5 Å) interproton-distance data^1,2. To obtain these distances, the ¹H-NMR spectrum must first be assigned using correlation and nuclear Overhauser effect (NOE) experiments to demonstrate through-bond (scalar) and through-space connectivities, respectively. Because the NOE is proportional to r^–6, distance information can then be derived. The increased resolution afforded by extending NMR experiments into a second dimension³ enables one to detect and interpret effects that would not be possible in one dimension owing to extensive spectral overlap and much reduced information. A number of small protein structures have previously been solved in this way^1,2. Extending this methodology to larger proteins, however, requires yet an additional improvement in resolution as overlap of cross-peaks in the two-dimensional (2D) NMR spectra present a major barrier to their unambiguous identification. One way of increasing the resolution is to extend the 2D-NMR experiments into a third dimension. We report here the applicability of three-dimensional NMR to macromolecules using the 46-residue protein al-purothionin as an example.