NMR N-15 RELAXATION AND STRUCTURAL STUDIES REVEAL SLOW CONFORMATIONALEXCHANGE IN BARSTAR C40 82A/

Barstar an 89-residue protein consisting of four helices and a three-s tranded parallel beta-sheet, is the intracellular inhibitor of the end oribonuclease barnase. Barstar C40/82A, a mutant in which the two cyst eine residues have been replaced by alanine, has been used as a pseudo wild-type in folding studies and in the crystal structure of the barn ase:barstar C40/82A complex. We have determined a high resolution solu tion structure of barstar C40/82A. The structures of barstar C40/82A a nd the wild-type are superimposable. A comparison with the crystal str ucture of the barnase:barstar C49/82A complex revealed subtle differen ces in the regions involved in the binding of barstar to barnase, Side -chain rotations of residues Asn33, Asp35 and Asp39 and a movement of the binding loop (Pro27-Glu32) towards the binding site of barnase fac ilitate the formation of interface hydrogen bonds and aromatic contact s in the complex. Extreme line broadening and missing signals in H-1-N -15 correlation spectra indicate substantial conformational exchange f or a large subset of residues. N-15 relaxation data at two magnetic fi eld strengths, 11.74 T and 14.10 T, were used to estimate exchange con tributions and to map the spectral density function at five frequencie s: 0, 50, 60, 450 and 540 MHz. Based on these results, model-fi-ee cal culations with the inclusion of estimated exchange contributions were used to derive order parameters and internal correlation times. The va lidity of this approach has been investigated with model-free calculat ions that incorporate longitudinal relaxation rates and heteronuclear H-1-N-15 NOE data only at 11.74 T and 14.10 T, The relaxation data sug gest substantial conformational exchange in regions of barstar C40/82A , Including the binding loop, the second and the third helices, and th e second and the third strands. Amide proton exchange experiments sugg est astable hydrogen bond network for all helices and sheets except th e third helix and the C-terminal of the second and the third strands, The combined results indicate a rigid body movement of the second heli x and twisting motions of the beta-sheet of barstar, which might be im portant for the interaction with barnase. (C) 1997 Academic Press Limi ted.