EFFECTS OF ION-BINDING ON THE BACKBONE DYNAMICS OF CALBINDIN-D9K DETERMINED BY N-15 NMR RELAXATION

The backbone dynamics of apo- and (Cd2+)1-calbindin D9k have been char acterized by N-15 nuclear magnetic resonance spectroscopy. Spin-lattic e and spin-spin relaxation rate constants and steady-state {H-1}-N-15 nuclear Overhauser effects were measured at a magnetic field strength of 11.74 T by two-dimensional, proton-detected heteronuclear NMR exper iments using N-15-enriched samples. There laxation parameters were ana lyzed using a model-free formalism that characterizes the dynamics of the N-H bond vectors in terms of generalized order parameters and effe ctive correlation times. The data for the apo and (Cd2+)1 states were compared to those for the (Ca2+)2 state [Kordel, J., Skelton, N. J., A kke, M., Palmer, A. G., & Chazin, W. J. (1 992) Biochemistry 31, 48 56 -4866] to ascertain the effects of ion ligation on the backbone dynami cs of calbindin D9k. The two binding loops respond differently to liga tion by metal ions: high-frequency (10(9)-10(12)s-1) fluctuations of t he N-terminal ion-binding loop are not affected by ion binding, wherea s residues G57, D58, G59, and E60 in the C-terminal ion-binding loop h ave significantly lower order parameters in the apo state than in the metal-bound states. The dynamical responses of the four helices to bin ding of ions are much smaller than that for the C-terminal binding loo p, with the strongest effect on helix III, which is located between th e linker loop and binding site II. Significant fluctuations on slower time scales also were detected in the unoccupied N-terminal ion-bindin g loop of the apo and (Cd2+)] states; the apparent rates were greater for the (Cd2+)1 state. These results on the dynamical response to ion binding in calbindin D9k provide insights into the molecular details o f the binding process and qualitative evidence for entropic contributi ons to the cooperative phenomenon of calcium binding for the pathway i n which the ion binds first in the C-terminal site.