Investigation of electrostatic interactions in two-stranded coiled-coils through residue shuffling.

The effects of electrostatic interactions on the stability of coiled-coils were investigated using the strategy of shuffling the sequence without changing the overall content of amino acid residues in the peptides. Shuffling the sequence provides peptides with thermodynamically similar unfolded states. Therefore, the unfolded state can be used as a universal reference state in comparing the thermodynamic properties of the folded coiled-coil structure of the peptides, while varying the configuration of ionized groups, that is, changing the types and number of potential electrostatic interactions. The relative stabilities of these states were determined by monitoring the temperature-induced folding/unfolding of the peptides in solutions with different pH and ionic strength by circular dichroism spectroscopy and scanning microcalorimetry. It was found that, in solutions with low ionic strength, ionic pairs contribute significantly to the stability of the coiled-coil conformation. The stability increases with an increase in the number of ionized groups in the peptide upon changing pH from acidic to neutral. In contrast, in the solutions with high ionic strength, the coiled-coil becomes less stable at neutral pH than at acidic pH. Most surprisingly, the increase in Gibbs energy of stabilization of the coiled-coil state with increasing pH at low ionic strength proceeds with a decrease in the enthalpy and entropy of unfolding. This observation can be explained only by hydration of ionized groups upon unfolding of coiled-coils which is associated with significant negative enthalpy and entropy effects.