CONFORMATIONAL TRANSITIONS WITHIN THE CALMODULIN-BINDING SITE OF BORDETELLA-PERTUSSIS ADENYLATE-CYCLASE STUDIED BY TIME-RESOLVED FLUORESCENCE OF TRP242 AND CIRCULAR-DICHROISM

The sequence situated around Trp242 in Bordetella pertussis adenylate cyclase, a bifunctional protein of 1706 amino acid residues, forms the core of the calmodulin-binding site. Peptides varying in size and in affinity for calmodulin, and preserving the same sequence around Trp24 2 were analyzed by time-resolved fluorescence spectroscopy. Their dyna mic properties were compared to those of the catalytic domain of B. pe rtussis adenylate cyclase corresponding to the first 400 amino acid re sidues of the protein and in which the Trp69 residue was replaced by P he. The heterogeneity of the fluorescence intensity decays of Trp242 i s likely due to the existence of conformers in equilibrium as is sugge sted by the effect of trifluoroethanol both on the secondary structure content and the lifetime distributions. Binding to calmodulin leads t o striking effects on the lifetime distribution profiles by selecting a major excited state population and therefore one major conformer. Tr p242 still presents some degree of rotational freedom in the complexes . The reduction of rotational freedom is more important for the shorte r peptides than for the longest one. A similar selection of one major conformer with the same lifetime was also observed for the Trp242 in t he mutant protein when bound to calmodulin, as in the complexes with t he peptides. We conclude that the site of interaction of B. pertussis adenylate cyclase with calmodulin has similar conformational flexibili ty as that evidenced in the isolated peptides. This property of the mo lecule allows a better adjustment of the enzyme upon interaction with calmodulin.