Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard ΔG, ΔH, and ΔS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20°C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes 'cold denaturation'.
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