Solution structures and thermodynamics of cis-trans X-Pro conformers of a novel single disulfide conopeptide

Abstract

The conopeptide Mo1853 (MW = 1853 Da) consists of 17 residues and a single disulfide bond. Structural studies using homonuclear solution NMR methods (2D 1H,1H DQF-COSY, TOCSY, NOESY and ROESY spectra) revealed that Mo1853 exists as two equally populated cis and trans X–Pro peptide bond conformers which are in slow exchange regime, compared to the chemical shift time scale. Temperature dependence of chemical shifts was measured and using coalescence temperature of two amide protons, the rate of exchange and the free energy of activation for the conformational exchange were determined to be 59 Hz and ≈ 67.2 kJ mol−1, respectively, at 318 K. Additional evidence for this conformational equilibrium was also observed as exchange correlation peaks in the 2D-NOESY and ROESY spectra. Tertiary structures of both the cis (PDB ID 8K3N) and trans (PDB ID 8K3M) conformers were determined using distance restraints, backbone dihedral angle restraints, the disulfide bond restraint and the cis or trans conformation of the X–Pro peptide bond. The trans conformer of Mo1853 is stabilized by hydrogen bonds while the cis conformer seems to be stabilized predominantly by hydrophobic interactions. This was further corroborated by the fact that at lower temperatures, the hydrophobic interactions became weaker reducing the population of the cis conformer with respect to that of the trans conformer. The cis and trans X–Pro peptide bond conformational exchange could be another means to enhance the structural variability of the conopeptides and could have significance in the synergistic functional response caused by the cone snail venom peptides.