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

Abstract

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 13C and 15N enriched protein species were determined. From these couplings the dihedral angles φ, ψ and χ1 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 T1, T2 and NOE build-up rates of 15N and 15C nuclei order parameters were obtained to describe the dynamic behaviour of protein molecular parts. Refined solution structures were obtained for ribonuclease T1, 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.