The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2'GMP have been studied by (15)N longitudinal and transverse relaxation experiments, combined with {(1)H, (15)H} NOE measurements. The intensity decay of individual amide cross peaks in a series of ((1)H, (15)N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain (15)N T(1) and T(2) relaxation times. These experimentally obtained values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559; 4559-4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.

Bile acids are generated in vivo from cholesterol in the liver, and they undergo an enterohepatic circulation involving the small intestine, liver, and kidney. To understand the molecular mechanism of this transportation, it is essential to gain insight into the three-dimensional (3D) structures of proteins involved in the bile acid recycling in free and complexed form and to compare them with homologous members of this protein family. Here we report the solution structure of the human ileal lipid-binding protein (ILBP) in free form and in complex with cholyltaurine. Both structures are compared with a previously published structure of the porcine ILBP-cholylglycine complex and with related lipid-binding proteins. Protein structures were determined in solution by using two-dimensional (2D)- and 3D-homo and heteronuclear NMR techniques, leading to an almost complete resonance assignment and a significant number of distance constraints for distance geometry and restrained molecular dynamics simulations. The identification of several intermolecular distance constraints unambiguously determines the cholyltaurine-binding site. The bile acid is deeply buried within ILBP with its flexible side-chain situated close to the fatty acid portal as entry region into the inner ILBP core. This binding mode differs significantly from the orientation of cholylglycine in porcine ILBP. A detailed analysis using the GRID/CPCA strategy reveals differences in favorable interactions between protein-binding sites and potential ligands. This characterization will allow for the rational design of potential inhibitors for this relevant system.

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel foimation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 ? 0.012 nm and 0.13 nm from the previously determined crystal structure. The sidechain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than Hisl14, with pK, values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pK, lower than 4.