STRUCTURE AND DYNAMICS OF BARNASE COMPLEXED WITH 3'-GMP STUDIED BY NMR-SPECTROSCOPY

The binding of 3'-GMP to the ribonuclease, barnase, has been studied u sing heteronuclear 2D and 3D NMR spectroscopy. The H-1 and N-15 NMR sp ectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D N OESY spectra have been used to identify inter- and intramolecular NOEs , and a solution structure for the barnase-3'-GMP complex has been cal culated. The position of the guanine ring of the ligand is reasonably well defined in the structures. The guanine ring forms hydrogen bonds with the NH protons of Ser57 and Arg59. These residues are located in a loop that is conserved among the microbial guanine-specific ribonucl eases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which h ave been shown to be involved in catalysis. The phosphate group of 3'- GMP is close to a number of positively charged residues that have also been shown to be important for activity. The position of the sugar mo iety of 3'-GMP is less well defined in the structures. Structures calc ulated for the complex could not simultaneously satisfy all the observ ed intermolecular NOEs for the sugar protons, suggesting that the suga r samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the cryst al structures of nucleotides bound to related ribonucleases. 3'-GMP bi nding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decre ase in the rates of hydrogen/deuterium exchange and aromatic ring rota tion in the active site of barnase upon ligand binding.