Investigations of peptide structural stability in vacuo
Kalapothakis, Jason Michael Drosos
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Gas-phase analytical techniques provide very valuable tools for tackling the structural complexity of macromolecular structures such as those encountered in biological systems. Conformational dynamics of polypeptides and polypeptide assemblies underlie most biological functionalities, yet great difficulties arise when investigating such phenomena with the well-established techniques of X-ray crystallography and NMR. In areas such as these ion mobility interfaced with mass spectrometry (IMMS) and molecular modelling can make a significant contribution. During an IMMS experiment analyte ions drift in a chamber filled with an inert gas; measurement of the transport properties of analyte ions under the influence of a weak electric field can lead to determination of the orientationally-averaged collision cross-section of all resolved ionic species. A comparison with cross-sections estimated for model molecular geometries can lead to structural assignments. Thus IMMS can be used effectively to separate gas-phase ions based on their conformation. The drift tube employed in the experiments described herein is thermally regulated, which also enables the determination of collision cross-sections over a range of temperatures, and can provide a view of temperature-dependent conformational dynamics over the experimental (low microsecond) timescale. Studies described herein employ IMMS and a gamut of other MS-based techniques, solution spectroscopy and – importantly – molecular mechanics simulations to assess a) conformational stability of isolated peptide ions, with a focus on small model peptides and proteins, especially the Trp cage miniprotein; and b) structural characteristics of oligomeric aggregates of an amyloidogenic peptide. The results obtained serve to clarify the factors which dominate the intrinsic stability of non-covalent structure in isolated peptides and peptide assemblies. Strong electrostatic interactions are found to play a pivotal role in determining the conformations of isolated proteins. Secondary structures held together by hydrogen bonding, such as helices, are stable in the absence of solvent, however gas-phase protein structures display loss of their hydrophobic cores. The absence of a polar solvent, “self-solvation” is by far the most potent force influencing the gas-phase configuration of these systems. Geometries that are more compact than the folded state observed in solution are routinely detected, indicating the existence of intrinsically stable compact non-native states in globular proteins, illuminating the nature of proteins’ ‘unfolded’ states.