BACKBONE DYNAMICS OF RIBONUCLEASE-T1 AND ITS COMPLEX WITH 2'GMP STUDIED BY 2-DIMENSIONAL HETERONUCLEAR NMR-SPECTROSCOPY

The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2'GMP have been studied by N-15 longitudinal an d transverse relaxation experiments, combined with {H-1,H-15} NOE meas urements. The intensity decay of individual amide cross peaks in a ser ies of(H-1,N-15)-HSQC spectra with appropriate relaxation periods (Kay ,L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay,L.E. et al. (1999 ) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain N-15 T-1 and T-2 relaxati on 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, Sec., 104, 4546-4559; 4559-4570). Th e microdynamical parameters accessible by this approach clearly indica te a correlation between the structural flexibility and the tertiary s tructure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The re sults obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.