37] Recognition and characterization of calmodulin-binding sequences in peptides and proteins

Abstract

Publisher Summary This chapter provides methods for the recognition and analysis of basic, amphiphilic calmodulin-binding regions in peptides and proteins. The first calmodulin-binding peptides were discovered through screening of peptide hormones for calmodulin-binding activity. Calmodulin also contains a hydrophobic patch that becomes more exposed upon calcium binding. Recent evidence has given credence to the premise that amphiphilic α helical peptides are good models for the calmodulin-binding site of target enzymes. A 27-residue peptide has been isolated from a CNBr digest of skeletal muscle myosin light chain kinase (MLCK), which appears to comprise this enzyme's calmodulin-binding domain. The helical wheel is a widely used qualitative method for locating potential calmodulin-binding sites within a protein sequence. A rapid method of quantitating the information presented on a helical wheel involves calculation of the average helical hydrophobic moment (μH), which is defined as the mean vector sum of the hydrophobicities of the residues in the helix. Binding of peptides to calmodulin can be determined in a number of ways that rely on monitoring changes in the physical and spectroscopic properties of calmodulin and/or peptides that accompany complex formation. Fluorescence spectroscopy is an excellent method for evaluating. It is also shown that peptides become more helical when they bind calmodulin and that this increase in helicity is readily measured by far-ultraviolet circular dichroism spectroscopy (CD).