NUCLEAR MAGNETIC RESONANCE--NEW METHODS AND MOLECULAR STRUCTURE DETERMINATION

已发展出新的方法来作共振分配

New approaches have been developed for making resonance assignments in larger proteins, up to about 25 kDa. The new methods do not rely on the small and frequently unresolvable homonuclear proton J coupling, but instead utilize relatively large and well resolved homo-and heteronuclear one-bond couplings. To reduce resonance overlap, it is necessary to spread the new spectra in three independent frequency dimensions. Using this new methodology, complete backbone resonance assignments were obtained for the protein calmodulin (16.7 kDa) in a very straightforward manner. Using an analogous approach, complete proton and carbon resonance assignments were made for the amino acid side chains of both calmodulin and interleukin-1- beta. A newly developed heteronuclear 3D NOESY experiment permits determination of a very large number of interproton distances, which form the basis of structure calculations, currently in progress. We have demonstrated for the first time that recording of four-dimensional NMR spectra is feasible, and can actually be very useful for assigning specific NOE interactions.