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43138-AC4
Characterization of a Peptide Model for Protein Unfolded States
The goal of this project has been to characterize the conformational properties of a peptide model for protein unfolded states. We, and others, have shown previously that common denaturants such as urea perturb the ensemble of states adopted by an unfolded protein, making denatured proteins poor models for unfolded proteins under folding conditions. In this project we are employing a host-guest peptide model. The host peptide is of sequence Ac-(Lys)2-(Ala)7-(Lys)2-NH2, with the central Ala being the guest residue site. This peptide model was chosen because it has a tendency towards alpha-helix formation, but is too short to form a stable helix. It is therefore a model for unfolded protein sequences that will fold into an alpha-helical conformation. We have studied the host peptide, plus peptides with Gly, Pro, Val, Ile, Leu, Phe, Ser, Thr, Asn and Gln guest residues. The conformational properties of these peptides under a variety of solution conditions have been determined using circular dichroism (CD) spectroscopy. We have found that the host peptide does indeed possess some alpha-helix character, which appears to be in equilibrium with the polyproline II (PII) helical conformation, plus other conformations. The various guest residues perturb this equilibrium in a variety of ways. Our most significant finding to date is that the well-established alpha-helix residue propensities are poor predictors of the conformational behavior of unfolded alpha-helices. A manuscript describing our findings is currently in preparation.
We have extended our studies to include steady state fluorescence resonance energy transfer (FRET) measurements. These experiments are progressing well and the data has proven interesting. Despite often dissimilar CD spectra for the host-guest peptides, they all have similar end-to-end distances. This result at first appears surpising. However, from polymer theory this would actually be the expected outcome. The peptides we are studying are all just a little longer than the “blob” length for polypeptides (~7-9 residues). The end-to-end distance for flexible peptides of the blob length should be approximately the same no matter what the sequences or average backbone conformational properties. We are finishing up these experiments and expect to publish the data soon.
