Is it biologically relevant to measure the structures of small peptides in the gas-phase?

Abstract

Recent developments in sample introduction of biologically relevant molecules have heralded a new era for gas-phase methods of structural determination. One of the biggest challenges is to relate gas-phase structures, often measured in the absence of water and counter ions, with in vivo biologically active structures. An advantage of gas-phase based techniques is that a given peptide can be analysed in a variety of different forms, for example, as a function of charge state, or with additional water molecules. Molecular modelling can provide insight into experimental findings and help elucidate the differences between structural forms. Combining experiment and theory provides a thorough interrogation of candidate conformations. Here two important naturally occurring peptide systems have been examined in detail and results are assessed in terms of their biological significance. The first of these is gonadotropin-releasing hormone (GnRH), a decapeptide which is the central regulator of the reproductive system in vertebrates. We have examined several naturally occurring variants of this peptide using Ion Mobility Mass Spectrometry and Electron Capture Dissociation (ECD) in conjunction with Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR-MS). Candidate conformations are modelled using the AMBER force field. Single amino acid changes, for example Gly6 → Ala6, or Ala6 → D-Ala6, have observable effects on the gas phase structure of GnRH. It has been shown that evolutionary primary sequence variations are key to the biological activity of GnRH, and it is thought that this is due to different binding affinities at target receptors. This work provides strong evidence that this activity is structurally based. The second system examined is the relationship between the quaternary structure and activity of two novel β-defensins. FT-ICR mass spectrometry has been employed to characterize di-sulphide bridging and dissociation based experiments utilised to investigate their structural core. Defr1, with five cysteines, exists as a covalently bound disulphide linked dimer; Defr1 Y5C with six cysteines also is observed as a dimer, but non-covalently bound, suggesting that this defensin has a tendency to aggregate. The activity of Defr1 is 10 times higher than that of Defr1 Y5C when tested against the pathogen Pseudomonas aeruginosa. The results from these studies could inform future design of novel GnRH type ligands and anti-microbial agents, and illustrate the power of gas-phase based techniques for solving peptide structures.