Detailed knowledge of the tertiary and quaternary structure of proteins and protein complexes is of immense importance in understanding their functionality. Similarly, variations in the conformational states of proteins form the underlying mechanisms behind many biomolecular processes, numerous of which are disease-related. Thus, the availability of reliable and accurate biophysical techniques that can provide detailed information concerning these issues is of paramount importance. Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) offers a unique opportunity to separate multi-component biomolecular entities and to measure the molecular mass and collision cross-section of individual components in a single, rapid (≤ 2 min) experiment, providing 3D-architectural information directly. Here we report a method of calibrating a commercially available electrospray ionisation (ESI)-travelling wave ion mobility spectrometry (TWIMS)–mass spectrometer using known cross-sectional areas determined for a range of biomolecules by conventional IMS-MS. Using this method of calibration, we have analysed a range of proteins of differing mass and 3D architecture in their native conformations by ESI-TWIMS-MS and found that the cross-sectional areas measured in this way compare extremely favourably with cross-sectional areas calculated using an in-house computing method based on Protein Data Bank NMR-derived co-ordinates. This not only provides a high degree of confidence in the calibration method, but also suggests that the gas phase ESI-TWIMS-MS measurements relate well to solution-based measurements derived from other biophysical techniques. In order to determine which instrumental parameters affect the ESI-TWIMS-MS cross-sectional area calibration, a systematic study of the parameters used to optimise TWIMS drift time separations has been carried out, observing the effect each parameter has on drift times and IMS resolution. Finally, the ESI-TWIMS-MS cross-sectional area calibration has been applied to the analysis of the amyloidogenic protein β₂-microglobulin and measurements for three co-populated conformational families, present under denaturing conditions, have been made: the folded, partially unfolded and unfolded states.

Dopamine (DA) and α‐synuclein (α‐SN) are two key molecules associated with Parkinson's disease (PD). We have identified a novel action of DA in the initial phase of α‐SN aggregation and demonstrate that DA induces α‐SN to form soluble, SDS‐resistant oligomers. The DA:α‐SN oligomeric species are not amyloidogenic as they do not react with thioflavin T and lack the typical amyloid fibril structures as visualized with electron microscopy. Circular dichroism studies indicate that in the presence of lipid membranes DA interacts with α‐SN, causing an alteration to the structure of the protein. Furthermore, DA inhibited the formation of iron‐induced α‐SN amyloidogenic aggregates, suggesting that DA acts as a dominant modulator of α‐SN aggregation. These observations support the paradigm emerging for other neurodegenerative diseases that the toxic species is represented by a soluble oligomer and not the insoluble fibril.

The Amyloid β peptide (Aβ) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu²⁺ ions can induce the de novo aggregation of the Aβ peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu²⁺ mediates the change from amyloid material toward Cu²⁺ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Aβ1−42 at neutral pH is governed by the Cu²⁺:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu²⁺ switching effect centered at equimolar Cu²⁺:peptide ratios. At sub-equimolar Cu²⁺:peptide molar ratios, Aβ1−42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu²⁺:peptide molar ratios, Aβ1−42 forms both small spherical oligomers approximately 10−20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu²⁺ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu²⁺ induced aggregates are toxic when compared to Aβ1−42 aged in the absence of Cu²⁺. Importantly, the formation of dityrosine crosslinked Aβ, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu²⁺ induced aggregates. These results define the role Cu²⁺ plays in modulating the aggregation state and toxicity of Aβ1−42.

Amelyoid-β peptide (Aβ) is a major causative agent responsible for Alzheimer's disease (AD). Aβ contains a high affinity metal binding site that modulates peptide aggregation and toxicity. Therefore, identifying molecules targeting this site represents a valid therapeutic strategy. To test this hypothesis, a range of L-PtCl ₂ (L = 1,10-phenanthroline derivatives) complexes were examined and shown to bind to Aβ, inhibit neurotoxicity and rescue Aβ-induced synaptotoxicity in mouse hippocampal slices. Coordination of the complexes to Aβ altered the chemical properties of the peptide inhibiting amyloid formation and the generation of reactive oxygen species. In comparison, the classic anticancer drug cisplatin did not affect any of the biochemical and cellular effects of Aβ. This implies that the planar aromatic 1,10-phenanthroline ligands L confer some specificity for Aβ onto the platinum complexes. The potent effect of the L-PtCl ₂ complexes identifies this class of compounds as therapeutic agents for AD.

Amyloid-beta peptide (Abeta) is pivotal to the pathogenesis of Alzheimer disease. Here we report the formation of a toxic Abeta-Cu2+ complex formed via a histidine-bridged dimer, as observed at Cu2+/peptide ratios of >0.6:1 by EPR spectroscopy. The toxicity of the Abeta-Cu2+ complex to cultured primary cortical neurons was attenuated when either the pi -or tau-nitrogen of the imidazole side chains of His were methylated, thereby inhibiting formation of the His bridge. Toxicity did not correlate with the ability to form amyloid or perturb the acyl-chain region of a lipid membrane as measured by diphenyl-1,3,5-hexatriene anisotropy, but did correlate with lipid peroxidation and dityrosine formation. 31P magic angle spinning solid-state NMR showed that Abeta and Abeta-Cu2+ complexes interacted at the surface of a lipid membrane. These findings indicate that the generation of the Abeta toxic species is modulated by the Cu2+ concentration and the ability to form an intermolecular His bridge.