Envisaging Structural Insight of a Terminally Protected Proline Dipeptide by Raman Spectroscopy and DFT Analyses

Envisaging Structural Insight of a Terminally Protected Proline Dipeptide by Raman Spectroscopy and DFT Analyses

Supriya Das, Uttam Pal, Moumita Chatterjee, Sumit Kumar Pramanik, Biswadip Banerji, Nakul Chandra Maiti

2016

Journal of Physical Chemistry A

The proline residue in a protein sequence generates constrains to its secondary structure as the associated torsion angles become a part of the heterocyclic ring. It becomes more significant when two consecutive proline residues link via amide linkage and produce additional configurational constrain to a protein’s folding and stability. In the current manuscript we have illustrated conformation preference of a novel dipeptide, (R)-tert-butyl 2-((S)-2-(methoxycarbonyl)pyrrolidine-1-carbonyl)pyrrolidine-1-carboxylate. The dipeptide crystallized in orthorhombic crystalline state and produced rod shaped macroscopic material. The analysis of the crystal coordinates showed dihedral angles (φ, ψ) of the interlinked amide groups as (72°, -147°) and the dihedral angles (φ, ψ) produced with the next carbonyl was (-68°, 151°) indicating polyglycine II (PGII) and polyproline II (PPII) like helix states at N- and C-terminals, respectively. These two states, PGII and PPII are mirror image like configuration and expected to produce similar vibration bands from the associated carbonyl groups. However, the unique atomic arrangement in the molecule produces three carbonyl groups and one of them was very specific being part of the main peptide linkage that connects both the pyrrolidine rings. The carbonyl group in the peptide bond exhibited Raman vibration frequency at ~1642 cm-1 and considered as signatory Raman marker band for the peptide bond linking two heterochiral proline residues. The carbonyl group (t-Boc) at the N-terminal of the peptide showed characteristic vibration at ~ 1685 cm-1 and the C-terminal carbonyl group as a part of the ester showed vibration signature at a significantly high frequency (1746 cm-1). Conformation analyses performed with DFT calculation depicted that the dipeptide was stabilized in vacuum with dihedral angles (72°, -154°) and (-72°, 151°) at N- and C-terminals respectively. Molecular dynamics (MD) simulation also showed that the peptide conformation having dihedral angles around (75°, -150°) and (-75°, 150°) at N- and C-terminals, respectively, was reasonably stable in water. Due to unique absence of the amide N-H, the peptide was ineffective to form any intramolecular hydrogen bonding. MD investigation, however, revealed intermolecular hydrogen bonding interaction with the water molecules leading to its stability in aqueous solution. Metadynamics simulation analysis of the dipeptide in water also supported the PGII-PPII like conformation at N- and C-terminals respectively as the energetically stable conformation among the other possible combinations of conformations. The possible electronic transitions along with the HOMO-LUMO analysis further depicted the stability of the dipeptide in water and their possible absorption pattern. Time-dependent density functional theory (TDDFT) analysis showed strong negative rotatory strength of the dipeptide around 210 nm in water and acetonitrile and, it could be the source of experimentally observed high-amplitude negative absorption in the circular dichroism (CD) spectra around 200-203 nm. The very weak positive band (signature) in the region at ~228 nm in CD spectra could also be correlated to the positive rotatory strength at 228 nm observed in ECD. To test the effect of such dipeptide on living cell, an MTT assay was performed and the result indicated no cytotoxic effect towards human hepatocellular carcinoma Hep G2 cancer cell lines.

http://pubs.acs.org/doi/abs/10.1021/acs.jpca.6b10017

J-815

Circular dichroism, Secondary structure, Biochemistry