Phenylalanine fluorescence studies of calcium binding to N-domain fragments of Paramecium calmodulin mutants show increased calcium affinity correlates with increased disorder.

Abstract

Calmodulin (CaM) is a ubiquitous, essential calcium-binding protein that regulates diverse protein targets in response to physiological calcium fluctuations. Most high-resolution structures of CaM-target complexes indicate that the two homologous domains of CaM are equivalent partners in target recognition. However, mutations between calcium-binding sites I and II in the N-domain of Paramecium calmodulin (PCaM) selectively affect calcium-dependent sodium currents. To understand these domain-specific effects, N-domain fragments (PCaM(1-75)) of six of these mutants were examined to determine whether energetics of calcium binding to sites I and II or conformational properties had been perturbed. These PCaM((1-75)) sequences naturally contain 5 Phe residues but no Tyr or Trp; calcium binding was monitored by observing the reduction in intrinsic phenylalanine fluorescence at 280 nm. To assess mutation-induced conformational changes, thermal denaturation of the apo PCaM((1-75)) sequences, and calcium-dependent changes in Stokes radii were determined. The free energy of calcium binding to each mutant was within 1 kcal/mole of the value for wild type and calcium reduced the R(s) of all of them. A striking trend was observed whereby mutants showing an increase in calcium affinity and R(s) had a concomitant decrease in thermal stability (by as much as 18 degrees C). Thus, mutations between the binding sites that increased disorder and reduced tertiary constraints in the apo state promoted calcium coordination. This finding underscores the complexity of the linkage between calcium binding and conformational change and the difficulty in predicting mutational effects.