Refined NMR solution structures of proteins using homo- and heteronuclear couplings, relaxation time measurements and relaxation matrix analysis

In order to compare high resolution crystal structures of proteins with the corresponding solution structures a detailed analysis of NMR parameters obtained for various proteins was carried out.As many NOE values as possible were transformed into distances using a relaxation matrix analysis.In addition homo-and heteronuclear 3J couplings from I3C and I5N enriched protein species were determined.From these couplings the dihedral angles $, and x1 were evaluated.It was possible to interpret the various 3J values in terms of either distinct dihedral angles or with a certain variance of angles or with an equilibrium of different rotameric states.The refined solution structures were obtained using the distance constraints together with the dihedral angle constraints in a distance geometry algorithm (DIANA program package).The resulting DG structure was the starting conformation of a subsequent molecular dynamics simulation.From a determination of relaxation times TI, T, and NOE build-up rates of "N and I3C nuclei order parameters were obtained to describe the dynamic behaviour of protein molecular parts.Refined solution structures were obtained for ribonuclease TI, a flavodoxin from D.vulgaris, fatty acid binding protein from bovine heart and for a heat shock transcription factor from tomato.In most cases the high resolution crystal structures differ only slightly from the refined solution structures.We have determined four possible vicinal coupling constants related to the dihedral angle 0 (LO) ' J-, J, , , JmFs and 'J&c,., and most of the vicinal coupling constants related to x, (11).The 'I data are analysed with respect to different models for the conformational dynamcis of the backbone and of the side 3 9