A Model for Circular Dichroism Monitored Dimerization and Calcium Binding in an EF-Hand Synthetic Peptide

Abstract

EF-hand peptides have been shown to bind calcium and dimerize to form an intact protein domain [Shaw, G.S., Hodges, R.S. & Sykes, B.D. (1990). Science, 249, 280–283]. A synthetic 33-residue EF-hand peptide with the sequence of carp parvalbumin CD site demonstrated a seven-fold increase in the apparent calcium dissociation constant with a eight-fold decrease in peptide concentration when fit to a single-site calcium-binding model. This observation is consistent with EF-hand dimerization. This paper describes a method to determine the dimerization dissociation constant and the calcium dissociation constants for both the monomer and dimer forms of this EF-hand peptide using circular dichroism techniques. By monitoring the increase in negative molar ellipticity at 222 nm with increasing peptide concentration under calcium-saturating conditions the dimerization dissociation constant for the synthetic parvalbumin CD site was determined to be 55.68±10.76 μM. Using the dimerization constant, the calcium dissociation constants for both the monomer and dimer forms of this peptide were determined by monitoring the change in ellipticity of peptide solutions on addition of increasing amounts of calcium. A fit of this data to a mathematical model that takes into account dimerization results in calcium dissociation constants of 421.3±21.56 and 47.06±6.72 μM for the monomer and dimer forms, respectively.