Calmodulin has been implicated in the regulation of a wide variety of calcium ion dependent cellular events in eukaryotic tissues. These processes are mediated by various enzymes the activities of which are regulated by interaction with calmodulin. The ultimate goal of this research is to understand the structural determinants for the interaction of calmodulin with these enzymes. The major problem in directly studying and interpreting calmodulin-enzyme interactions is that the interacting enzyme is usually a complex macromolecule of undetermined structure. Therefore this research project will focus on understanding the molecular mechanisms of these interactions with the aid of newly designed model peptides that will have calmodulin affinities as high as the calmodulin-dependent enzymes. The results will contribute to the enhancement of our understanding of the mechanism of strong interactions between two protein surfaces. The advantages of using small synthetic peptides are that: (i) the primary structure is precisely known, (ii) predictions can be made about their secondary structure forming potential and (iii) conformation in different environments can be studied and more readily interpreted than in the case of a complex enzyme. The major specific aims are to determine (i) the precise structural features in a peptide required for high affinity binding to calmodulin, (ii) what conformation an interacting peptide assumes when bound to calmodulin and (iii) the structure of the complementary calmodulin surface. It would then be possible to identify regions, in calmodulin binding enzymes, that show the potential of folding into such conformation under the conditions provided by the interacting calmodulin surface. Each peptide will be designed to have a single tryptophan residue that will provide an unambiguous (since calmodulin does not contain tryptophan) fluorescence spectroscopic probe to monitor interactions of different peptide regions with calmodulin. In order to correlate peptide structure with calmodulin binding affinity, the dissociation constants of the calmodulin-peptide complexes will be determined by monitoring changes in fluorescence intensity and anisotropy. Fluorescence energy transfer experiments will be used to determine conformational changes in the peptide upon calmodulin binding. Changes in the secondary structure will be further studied by circular dichroism. The nature of the interacting surface on calmodulin will be studied (i) by introducing environment sensitive fluorescent probes and (ii) by studying interactions of specific calmodulin fragments with the peptides.
| Sanyal, G; Richard, L M; Carraway 3rd, K L et al. (1988) Binding of amphiphilic peptides to a carboxy-terminal tryptic fragment of calmodulin. Biochemistry 27:6229-36 |
