Free Radical-Initiated Unfolding of Peptide Secondary Structure Elements

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The present thesis investigates the effect of radical formation on peptide secondary structure elements, and analyses the changes such radical formations can induce. To determine if the pro-L hydrogen on Cα of the glycyl residues are more prone to hydrogen abstraction by •OH than the respective hydrogen in Ala, the pre-reaction van der Waals complexes, transition states, and post-reaction van der Waals complexes of H abstraction from Ala and Gly were computed. To evaluate the conformational effect of H abstraction, different conformers (βL, γL, γD, αL and αD) of these residues were studied. These calculations were carried out at the MPWKCIS1K/6-311++G(3df,2p)//BHandHLYP/6- 311+G(d,p) level of theory. To study the effect of hydrogen abstraction on peptide secondary structures, model pentapetides were also studied. Helical unfolding (as shown in Figure 1) was investigated by computing the thermodynamic functions of the radical-initiated unfolding of a helix. A hydrogen atom was extracted from the Cα and amide nitrogen of Gly3 , and the Cα, Cβ and amide nitrogen of Ala3 , of the respective G5 (N-Ac-GGGGG-NH2) and A5 (N-Ac-AAAAANH2) homo-peptides. The HO• , HO2 • and O2 -• radicals were used in each case, and the thermodynamic functions were computed using the B3LYP density functional. The changes in potential energy, standard enthalpy, Gibbs free energy and entropy during these reactions were computed with G5 and A5 in the 310-helical and fully-extended conformations. These computations were carried out in the gas phase and the effect of solvent was mimicked with the C-PCM implicit water model. Figure 1. A schematic representation of the radical-initiated unfolding of an amino acid residue. To enable the effects of Cα-centered radicals to be studied in longer peptides and proteins over greater time intervals with molecular dynamics (MD), force field parameters for the Cαcentered Ala radical were developed for use with the OPLS force field. This was done by minimizing the sum of squares deviation between the quantum chemical and OPLS-AA energy hypersurfaces. These parameters were used to determine the effect of the Cα-centered Ala radical on the structure of a hepta-alanyl peptide in molecular dynamics simulations. A dramatic change in conformation is observed in the Gly and Ala conformers after converting to Gly• and Ala• , respectively, and this change can be monitored along the minimal energy pathway by computing the intrinsic reaction coordinate for the conformers of each residue. The βL conformer of Gly and Ala form the lowest-lying transition states, whereas the side-chain of Ala strongly destabilizes the α conformers compared to the γ conformers. The energies of the α to γ transition in Gly are more similar than those of Ala. This effect shown in Ala could inhibit the abstraction of hydrogen from the chiral amino acid residues in the helices. The energy of a subsequent hydrogen abstraction reactions between Ala• and Gly• and H2O2 remains approximately 90 kJ mol-1 below the entrance level of the •OH reaction, indicating that the •OH radical can initiate an α to β transition in an amino acid. However, a molecule such as H2O2 must provide the hydrogen atom necessary to reform the Gly and Ala residues. As shown in the G5 and A5 peptides, hydrogen abstraction is the most favorable at the Cα, followed by the Cβ , then amide nitrogen. The secondary structure has a strong influence on the bond dissociation energy of the H-Cα, but a negligible effect on the dissociation energy of the H-CH2 and H-N bonds. The HO• radical is the strongest hydrogen abstractor, followed by HO2 • and finally O2 -• . Secondary structure elements, like H-bonds in the 310-helix, protect the peptide from radical attack by hindering the potential electron delocalization at the Cα which is present when the peptide is in the extended conformation. The Cα-centered pentapeptide radicals have a significantly higher propensity to unfold than the closed shell pentapeptides. Furthermore, only the HO• radical can initiate the unfolding of the pentapeptides to an extended pentapeptide radical, and the unfolding of the Cα-centered G5 is more favorable than the unfolding of the Cα-centered A5. A negligible sum-of-squares energy deviation was observed in the stretching parameters, and the newly-developed OPLS-AA torsional parameters showed a good-agreement with the LMP2/cc-pVTZ(-f) hypersurface. The MD simulations showed planar conformations of the residue with the radical on its alpha carbon (Alr) are preferred and these conformations increase the formation of γ- α- and π-turn structures depending on the position in the turn occupied by the Alr residue. Higher-ordered structures are destabilized by Alr except when this residue occupies position “i + 1” of the 310-helix. These results offer new insight in to the protein-misfolding mechanisms initiated by Habstraction from the Cα of peptide and protein residues.

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