The effect of substituting hydrophobic residues in heptad repeats of the de novo lanthanide-binding coiled coil polypeptide MB1–2 on the thermodynamic stability of the resulting Tb-peptide complex was examined. The analog complex seems to have a trimeric structure equivalent to that formed by MB1–2, but with increased stability.
ABSTRACT
A de novo lanthanide-binding coiled-coil polypeptide (MB1–2) was previously reported to self-assemble into a trimeric complex upon addition of Tb3+ with a micromolar range dissociation constant. This study examines the effect of substitution of hydrophobic residues in heptad repeats of MB1–2 on the thermodynamic stability of the resulting Tb-peptide complex. Substitution of isoleucine to norleucine in each heptad repeat was assessed considering the greater accessible surface area of the latter and predicted increased hydrophobic interaction. Job’s method of continuous variation using circular dichroism spectroscopy suggests a trimeric structure for the analog complex equivalent to that formed by MB1–2. The dissociation constant and CD spectra suggest that complex formation in the analog is more favorable as a result of ligand preorganization. In addition, thermal denaturation suggests greater stability of the Tb-MB1–2 Nle complex in comparison to the parent Tb-MB1–2. These results indicate improved stability of the complex class can be achieved through heptad repeat amino acid substitutions that increase peptide interchain interaction.
