We present the high-resolution solution structure and 13C assignments of wild-type barstar, an 89 amino acid residue polypeptide inhibitor of barnase, derived from heteronuclear NMR techniques. These were obtained from measurements on unlabeled, uniformly 15N- and 13C/15N-labeled, and 10% 13C-labeled barstar samples that have both cysteines (at positions 40 and 82) fully reduced. In total, 30 structures were calculated by hybrid distance geometry-dynamical simulated annealing calculations. The atomic rms distribution about the mean coordinate positions is 0.42 A for all backbone atoms and 0.90 A for all atoms. The structure is composed of three parallel alpha-helices packed against a three-stranded parallel beta-sheet. A more poorly defined helix links the second beta-strand and the third major alpha-helix. The loop involved in binding barnase is extremely well defined and held rigidly by interactions from the main body of the protein to both ends and the middle of the loop. This structure will be used to aid protein engineering studies currently taking place on the free and bound states of barstar and barnase.

Only very modest Ca(2+)-induced changes are observed in the structure of calcyclin, in sharp contrast to the domain-opening that occurs in calmodulin and related Ca(2+)-sensor proteins. Thus, calcyclin, and by inference other members of the S100 family, must have a different mode for transducing Ca2+ signals and recognizing target proteins. This proposal raises significant questions concerning the purported roles of S100 proteins as Ca2+ sensors.

The binding of 3'-GMP to the ribonuclease, barnase, has been studied using heteronuclear 2D and 3D NMR spectroscopy. The 1H and 15N NMR spectra of barnase complexed with 3'-GMP have been assigned. 2D and 3D NOESY spectra have been used to identify inter- and intramolecular NOEs, and a solution structure for the barnase-3'-GMP complex has been calculated. 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 ribonucleases. The 2'-hydroxyl of 3'-GMP is close to His102 and Glu73, which have 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 moiety of 3'-GMP is less well defined in the structures. Structures calculated for the complex could not simultaneously satisfy all the observed intermolecular NOEs for the sugar protons, suggesting that the sugar samples several conformations when bound to barnase. The binding of 3'-GMP to barnase in solution is similar to that observed in the crystal structures of nucleotides bound to related ribonucleases. 3'-GMP binding causes no major conformational change in barnase. In contrast to the small structural changes that occur, there is a significant decrease in the rates of hydrogen/deuterium exchange and aromatic ring rotation in the active site of barnase upon ligand binding.

Barstar, a polypeptide inhibitor of ribonucleases, has been studied by 2D and 3D NMR techniques using uniformly '5N-labeled protein. Backbone ("NH-C,H-CflH) resonances were assigned for all but 5 of the 89 residues. Dihedral angle and deuterium exchange studies were used in conjunction with medium range inter-proton NOES to characterize the secondary structure of barstar. The protein is composed of four a-helices and three short stretches of extended strand. By further analysis of the NOE data three of the helices were found to be parallel to each other with the single disulphide bond linking the second and fourth helices at their C-terminal ends.

The extracellular ribonuclease from Bucrllus amylohquifacwns, barnase. forms a tightly-bound one-to-one complex with its intracellular inhibitor barstar. The barstar binding site on barnase was charactemed by comparing the differences m the chemical shift and hydrogen-deuterium exchange rates between free and bound barnase Chemical shift assignments of bamase in the complex with barstar were determined from 3D NOESY-HMQC and TOCSY-HMQC spectra of a complex that had been prepared with uniformly 'SN-labelled barnase and unlabelled barstar. Hydrogen exchange rates were obtained from an analysis of a series of ["NIHMQC spectra of a sample prepared in the same manner exchanged into DZO. The largest changes in either chemical shift or hydrogendeuterium exchange rate are observed for residues located in the active-site and substrate binding loops indicating that barstar inhibits barnase activity by sterically blocking the active site.Barnase-barstar complex: NMR asslgment