Folding of the Transmembrane 25-Residues Influenza A M2 and Ala-25 Peptides

The correct balance between hydrophobic London dispersion (LD) and peptide hydrogen bonding interactions must be attained for proteins to fold correctly. To investigate these important contributors we sought a comparison of the influenza A transmembrane M2 protein (M2TM) 25-residues monomer and the 25-Ala (Ala25) peptide, used as reference since alanine is the only amino acid forming a standard peptide helix which is stabilized by the backbone peptide hydrogen bonding interactions. Folding molecular dynamics (MD) simulations were performed ing the AMBER99SB-STAR-ILDN force field in trifluoroethanol (TFE) as a membrane mimetic, to study the α-helical stability of M2TM and Ala25 peptides. It was shown that M2TM peptide did not form a single stable α-helix compared to Ala25. Instead appears to be dynamic in nature and quickly inter-converts between an ensemble of various folded helical structures having the highest thermal stability to the N-terminal compared to Ala25. Circular dichroism (CD) experiments confirm the stability of the α-helical M2TM. DFT calculations results revealed an extra stabilization for the folding of M2TM from b-strand to the α-helix compared to Ala25, due to forces that can't be described from a force field. On a technical level, calculations using D95(d,p) single point at a ONIOM (6-31G,3-21G) minimized geometry, in which the backbone is calculated with 6-31G and alkyl side chains with 3-21G, produced an energy differential for M2TM comparable with full D95(d,p). Natural bond orbital (NBO) and quantum theory of atoms in molecules (QTAIM) calculations were applied to investigate the relative contribution of N-H∙∙∙O as compared to C-H∙∙∙O hydrogen bonding interactions in the M2TM which included 17 lipophilic residues; 26 CH∙∙∙O interactions were identified, as compared to 22 NH∙∙∙O H-bonds. The calculations suggested that CH∙∙∙O hydrogen bonds, although individually weaker, have a cumulative effect that cannot be ignored and may contribute as much as half of the total interaction energy when compared to NH∙∙∙O to the stabilization of the folded α-helix in M2TM compared to Ala25.