A kinetic method for detecting intramolecular peptide H-bonds

The current method for detection of peptide hydrogen bond (PHB) formation places charge donors/acceptors (D/A) at N-/C-termini of the peptides involved in the putative PHB. The efficiency of the charge...

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

<p>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.</p>

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

<p>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.</p>

Dual self-assembly of supramolecular peptide nanotubes to provide stabilisation in water

Self-assembling peptides have the ability to spontaneously aggregate into large ordered structures. The reversibility of the peptide hydrogen bonded supramolecular assembly make them tunable to a host of different applications, although it leaves them highly dynamic and prone to disassembly at the low concentration needed for biological applications. Here we demonstrate that a secondary hydrophobic interaction, near the peptide core, can stabilise the highly dynamic peptide bonds, without losing the vital solubility of the systems in aqueous conditions. This hierarchical self-assembly process can be used to stabilise a range of different β-sheet hydrogen bonded architectures.

Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

Expression, Purification, and Structural Characterization of CfrA, a Putative Iron Transporter from Campylobacter jejuni

ABSTRACT The gene for the Campylobacter ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen Campylobacter jejuni, was cloned, and the membrane protein was expressed in Escherichia coli, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a β-sheet protein (∼44% β-sheet and ∼10% α-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to 2H2O and yet is resistant to thermal denaturation at temperatures up to 95°C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane β-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the E. coli enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.

Asymptotic Strength Limit of Hydrogen Bond Assemblies in Proteins at Vanishing Pulling Rates

ABSTRACTExperimental and computational studies on mechanical unfolding of proteins suggest that rupture forces approach a limiting value of a few hundred pN at vanishing pulling velocities. We develop a fracture mechanics based theoretical framework that considers the free energy competition between entropic elasticity of polypeptide chains and rupture of peptide hydrogen bonds, which we use here to provide an explanation for the intrinsic strength limit of proteins. Our analysis predicts that individual protein domains stabilized by hydrogen bonds can not exhibit rupture forces larger than approximately ≈200 pN, regardless of the presence of a large number of hydrogen bonds. This result explains a wide range of experimental and computational observations.