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 steady-state and time-resolved changes in the fluorescence associated with (1) the unique tyrosine (Tyr139) located in calcium binding loop IV or (2) N-(1-pyrenyl)-maleimide (PM) or 4-(iodoacetamido)salicylic acid (IASA) covalently attached to Cys27 present in calcium binding loop I. These fluorophores permit the measurement of calcium-dependent changes in (i) the solvent accessibility and rotational dynamics associated with calcium binding loops I and IV and (ii) the hydrodynamic properties of the entire protein. Specific nitration of the unique tyrosine (Tyr139) in calcium binding loop IV permits the use of fluorescence resonance energy transfer to measure both the average spatial separation and distance heterogeneity between Cys27 and Tyr139, providing a direct measurement of the conformational flexibility of the central helix. Upon calcium binding, (i) the solvent accessibility and rotational dynamics of both PM and IASA (covalently bound to Cys27) and Tyr139 increase, (ii) overall protein rotational motion decreases, (iii) the average separation between the chromophores at Cys27 and nitrotyrosine 139 decreases, and (iv) the conformational flexibility associated with the central helix decreases. Therefore, upon calcium binding, the central helix becomes more extended and rigid, while the globular domains adopt a more open tertiary conformation that brings Cys27 and Tyr139 into closer proximity. This calcium-dependent structural change functions to expose the hydrophobic binding sites located within the globular domains, and to enhance the probability of binding target sequences through a reduction in conformational heterogeneity.