We are using both gas and solution phase methodologies to examine non-covalent interactions between proteins and their binding partners. We have established methods that maintain protein ligand complexes in the gas phase and we probe the strength of association of these complexes by forcing them to dissociate using a variety of techniques. It is important to establish how robust are these tools given the ranges of energies put in to the complex prior to detection.

B-Defensins are an extremely important component of the innate defense mechanism of all mammalian species. They are cationic, amphipathic peptides, 30-45 amino acids in length, with exceptionally low sequence homology, save for 5 or 6 cysteines. Their activity is presumed related to a common disulphide bridging pattern, which in turn contributes to a common B-sheet motif.

We have examined 4 novel B-defensins using FT-ICR, have been able to unambiguously define the number of cross-linked cysteine resides in a given protein and have identified for the first time by mass spectrometry two B-defensin dimers. Our preliminary investigations have focussed on two recently discovered defensins which contain only 5 cysteine residues (DEFR1 and DEFB107). DEFR1 shows strong activity against Burkholderia cepacia, whereas the DEFB107 is completely inactive against bacteria.

We have examined a more typical B-defensin, DEFB2 with 6 cysteines and 3 intra-disulphide bonds. Our findings indicate that DEFR1 is present as a covalently bound dimer, due to an inter molecular disulphide bond between the ‘spare’ cysteine on each monomer residue. In contrast DEFB107 has its free cysteine capped with glutathione. This suggests a limit to the cysteine-bridging motif which defines the activity of these peptides.

We are currently using several methods to investigate these peptides. This work is extremely collaborative, we work alongside Dr. Dominic Campopiano (Edinburgh Chemistry), Dr. Julia Dorin (MRC-Human Genetics Unit), and Professor John Govan, (Edinburgh Medical School).

GHRH (gonatropin hormone releasing hormone) is 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 FTMS. Much of this work is performed in collaboration with Professor Bob Millar (MRC Human Reproductive Sciences Unit) and Professor Mike Bowers (UCSB).

Candidate conformations are modelled using the AMBER force field. We find that single amino acid changes, for example Gly⁶ to L-Trp⁶, or Gly⁶ to D-Trp⁶, have marked effects on the gas phase structure of LHRH. These differences change the fragmentation channels open to the molecule on ECD, since different intra-molecular bonds are formed and this is confirmed by modelled structures.

Hydration studies (performed in the Bowers group) using the mammalian form of LHRH, show a strong preference for binding the first water molecule with a measured -?H^o = 13.1 kcal mol^-1.

Complementary molecular modelling supports this and shows a distinct binding pocket for the first water centred on Arg8. Molecular dynamics show that this first water locks the quasi-circular conformation of the molecule.

However, calculations have also revealed a dramatic structural change on binding 3-4 water molecules. Essentially both on hydration, and on replacing Gly with more bulky amino acids, significant shifts are apparent in the confirmation of the polypeptide backbone.

A given peak in a mass spectrum may be due to one ion M⁺ with several different structures. The velocity with which this ion travels through a cell containing a buffer gas (commonly helium) under the influence of a weak electric field, depends on the ions collision cross section with the buffer gas (averaged over all possible orientations of the ion). Experimental cross sections for various systems can be found here.

A more extended isomer of M⁺ will take longer to traverse the drift cell (undergoing more collisions with the buffer gas) than a compact form, and a detector placed after the cell will record an arrival time distribution (ATD) which reflects these different drift times. This technique is know as Ion Chromatography (IC), and may be considered as the gas phase analogue of electrophoresis.

The arrival time of a given injected ion at a detector placed after the drift cell, can be used in combination with theoretical analysis to determine its structure. The simplest approximation for this is to determine the collision integral for a “projection” or “shadow” of the ion colliding with hard sphere buffer gas atoms.

A common approach is to generate several “shadow structures” via molecular modelling and compute expected arrival times for comparison with those experimentally determined. We are presently constructing an Ion Mobility Mass Spectrometer which will integrate a Micromass Q-Tof with a drift cell.

We use several dissociation techniques including, collision induced dissociation, in source dissociation, and Electron Capture dissociation (performed using the FT-ICR within the SIRCAMS facility).

Other Techniques:  Sonic Spray and Molecular Modelling