UV Raman Spatially Resolved Melting Dynamics of Isotopically Labeled Polyalanyl Peptide: Slow α-Helix Melting Follows 310-Helices and π-Bulges Premelting

Abstract

We used UV resonance Raman (UVRR) to examine the spatial dependence of the T-jump secondary structure relaxation of an isotopically labeled 21-residue mainly Ala peptide, AdP. The AdP penultimate Ala residues were perdeuterated, leaving the central residues hydrogenated, to allow separate monitoring of melting of the middle versus the end peptide bonds. For 5 to 30 °C T-jumps, the central peptide bonds show a ∼2-fold slower relaxation time (189 ± 31 ns) than do the exterior peptide bonds (97 ± 15 ns). In contrast, for a 20 to 40 °C T-jump, the central peptide bond relaxation appears to be faster (56 ± 6 ns) than that of the penultimate peptide bonds (131 ± 46 ns). We show that, if the data are modeled as a two-state transition, we find that only exterior peptide bonds show anti-Arrhenius folding behavior; the middle peptide bonds show both normal Arrhenius-like folding and unfolding. This anti-Arrhenius behavior results from the involvement of π-bulges/helices and 3₁₀-helix states in the melting. The unusual temperature dependence of the (un)folding rates of the interior and exterior peptide bonds is due to the different relative (un)folding rates of 3₁₀-helices, α-helices, and π-bulges/helices. Pure α-helix unfolding rates are ∼12-fold slower (∼1 μs) than that of π-bulges and 3₁₀-helices. In addition, we also find that the α-helix is most stable at the AdP N-terminus where eight consecutive Ala occur, whereas the three hydrophilic Arg located in the middle and at the C-terminus destabilize the α-helix in these regions and induce defects such as π-bulges and 3₁₀-helices.