Purification and characterization of an expressed peptide representing the regulatory domain of the gamma subunit of phosphorylase kinase.

The C-terminal 110 residues of the gamma-subunit of phosphorylase kinase have been termed the regulatory domain and contain two calmodulin-binding subdomains. In order to structurally characterize this region of the gamma-subunit, a bacterial expression system was developed to produce a 5-residue sequence (residues 302-377 of the gamma-subunit) from within the regulatory domain that contains the calmodulin-binding and pseudo substrate subdomains. The pMALc2 expression vector was employed which produces a fusion protein containing maltose-binding protein and the gamma-subunit peptide termed PhKpep. The bacterial expression system produced nanomolar amounts of the pure PhKpep peptide after purification of the fusion protein using amylase resin, cleavage with factory Xa, and high-performance liquid chromatography purification. PhKpep was characterized by N-terminal sequence analysis and ion spray mass spectrometry. PhKpep was shown to bind calmodulin with 1:1 stoichiometry in a calcium-dependent manner. The PhKpep peptide had a high affinity for calmodulin as expected from previous studies performed on constituent calmodulin-binding subdomains of PhKpep. The KD was determined to be in the subnanomolar range as determined by fluorescence anisotropy using carload-labeled T146C calmodulin. This peptide should be useful in a variety of structural studies aimed at understanding the molecular interactions of the gamma-subunit with calmodulin.