RESOLUTION OF STRUCTURAL-CHANGES ASSOCIATED WITH CALCIUM ACTIVATION OF CALMODULIN USING FREQUENCY-DOMAIN FLUORESCENCE SPECTROSCOPY

Structural changes associated with the calcium-dependent activation of wheat germ calmodulin (CaM) were assessed through measurements of ste ady-state and time-resolved changes in the fluorescence associated wit h (1) the unique tyrosine (Tyr(139)) located in calcium binding loop I V or (2) N-(1-pyrenyl)maleimide (PM) or 4-(iodoacetamido)salicylic aci d (IASA) covalently attached to Cys(27) present in calcium binding loo p I. These fluorophores permit the measurement of calcium-dependent ch anges in (i) the solvent accessibility and rotational dynamics associa ted with calcium binding loops I and IV and (ii) the hydrodynamic prop erties of the entire protein. Specific nitration of the unique tyrosin e (Tyr(139)) in calcium binding loop IV permits the use of fluorescenc e resonance energy transfer to measure both the average spatial separa tion and distance heterogeneity between Cys(27) and Tyr(139), providin g a direct measurement of the conformational flexibility of the centra l helix. Upon calcium binding, (i) the solvent accessibility and rotat ional dynamics of both PM and IASA (covalently bound to Cys(27)) and T yr(139) increase, (ii) overall protein rotational motion decreases, (i ii) the average separation between the chromophores at Cys(27) and nit rotyrosine 139 decreases, and (iv) the conformational flexibility asso ciated with the central helix decreases. Therefore, upon calcium bindi ng, the central helix becomes more extended and rigid, while the globu lar domains adopt a more open tertiary conformation that brings Cys(27 ) and Tyr(139) into closer proximity. This calcium-dependent structura l change functions to expose the hydrophobic binding sites located wit hin the globular domains, and to enhance the probability of binding ta rget sequences through a reduction in conformational heterogeneity.