Structural characterization of intrinsically disordered proteins (IDPs) has been a major challenge in the field of protein science due to limited capabilities to obtain full-length high-resolution structures. Native ESI-MS with top-down MS was utilized to obtain structural features of protein-ligand binding for the Parkinson's disease-related protein, α-synuclein (αSyn), which is natively unstructured. Binding of heavy metals has been implicated in the accelerated formation of αSyn aggregation. Using high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, native top-down MS with various fragmentation methods, including electron capture dissociation (ECD), collisional activated dissociation (CAD), and multistage tandem MS (MS), deduced the binding sites of cobalt and manganese to the C-terminal region of the protein. Ion mobility MS (IM-MS) revealed a collapse toward compacted states of αSyn upon metal binding. The combination of native top-down MS and IM-MS provides structural information of protein-ligand interactions for intrinsically disordered proteins. Graphical Abstract ᅟ.

Regulation of amyloid-β (Aβ) aggregation by metal ions and proteins is essential for understanding the pathology of Alzheimer's disease (AD). Human serum albumin (HSA), a regulator of metal and protein transportation, can modulate metal-Aβ interactions and Aβ aggregation in human fluid; however, the molecular mechanisms for such activities remain unclear. Herein, we report the molecular-level complexation between Zn(II), Cu(II), Aβ, and HSA, which is able to alter the aggregation and cytotoxicity of Aβ peptides and induce their cellular transportation. In addition, a single Aβ monomer-bound HSA is observed with the structural change of Aβ from a random coil to an α-helix. Small-angle X-ray scattering (SAXS) studies indicate that Aβ-HSA complexation causes no structural variation of HSA in solution. Conversely, ion mobility mass spectrometry (IM-MS) results present that Aβ prevents the shrinkage of the V-shaped groove of HSA in the gas phase. Consequently, for the first time, HSA is demonstrated to predominantly capture a single Aβ monomer at the groove using the phase transfer of a protein heterodimer from solution to the gas phase. Moreover, HSA sequesters Zn(II) and Cu(II) from Aβ while maintaining Aβ-HSA interaction. Therefore, HSA is capable of controlling metal-free and metal-bound Aβ aggregation and aiding the cellular transportation of Aβ via Aβ-HSA complexation. The overall results and observations regarding HSA, Aβ, and metal ions advance our knowledge of how protein-protein interactions associated with Aβ and metal ions could be linked to AD pathogenesis.

α-Synuclein (αSyn) is an intrinsically disordered protein, the aggregation of which is highly related to the pathology of diverse α-synucleinopathies. Various hard divalent metal cations have been shown to affect αSyn aggregation. Especially, Ca2+ is suggested to be a crucial ion due to its physiological relevance to α-synucleinopathies. However, the molecular origin of αSyn aggregation mediated by the metal ions is not fully elucidated. In this study, we revealed that hard divalent metal ions had almost identical influences on αSyn aggregation. Based on these similarities, the molecular role of Ca2+ was investigated as a representative metal ion. Herein, we demonstrated that binding of multiple Ca2+ ions induces structural transition of αSyn monomers to extended conformations, which promotes rapid αSyn fibrillation. Additionally, we observed that Ca2+ induced further interfibrillar aggregation via electrostatic and hydrophobic interactions. Our results from multiple biophysical methods, including ion mobility-mass spectrometry (IM-MS), synchrotron small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), provide detailed information on the structural change of αSyn and the aggregation process mediated by Ca2+. Overall, our study would be valuable for understanding the influence of Ca2+ on the aggregation of αSyn during the pathogenesis of α-synucleinopathies.

Structural variation of α-synuclein (αSyn) fibrils has been linked to the diverse etiologies of synucleinopathies. However, little is known about what specific mechanism provides αSyn fibrils with pathologic features. Herein, we demonstrate Cu(II)-based supramolecular approach for unraveling the formation process of pathogenic αSyn fibrils and its application in a neurotoxic mechanism study. The conformation of αSyn monomer was strained by macrochelation with Cu(II), thereby disrupting the fibril elongation while promoting its nucleation. This non-canonical process formed shortened, β-sheet enriched αSyn fibrils (<0.2 μm) that were rapidly transmitted and accumulated to neuronal cells, causing neuronal cell death, in sharp contrast to typical αSyn fibrils (ca. 1 μm). Our approach provided the supramolecular basis for the formation of pathogenic fibrils through physiological factors, such as brain Cu(II).

Advanced understanding of Alzheimer's disease (AD) and several tauopathies over the past decades indicates the pathological importance of tau aggregation in these diseases. Herein, we demonstrated that adenosine triphosphate (ATP), a highly charged anionic molecule abundant in the cytosol of cells, catalyses tau fibrillation via supramolecular complexation with basic residues of tau. Our results showed that ATP attracts multiple lysine residues of four-repeat domain of tau (K18), thereby immediately forming dimers which convert to nuclei to accelerate fibril elongation. However, ATP was not directly incorporated in the K18 fibrils suggesting a catalytic role of ATP in K18 fibrillation. We also characterized the correlation between ATP dyshomeostasis and tau aggregation in the cellular environment. Our multiple biophysical approaches, including native mass spectrometry (MS), small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulation, provided insights into the molecular-level influence of ATP on the structural change and fibrillation of tau. File list (2) download file view on ChemRxiv 200314_TauATP_Preprint.pdf (1.62 MiB) download file view on ChemRxiv 200314_SI_TauATP_Preprint.pdf (1.47 MiB)

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Flexible structures of intrinsically disordered proteins (IDPs) are crucial for versatile functions in living organisms, which involve interaction with diverse partners. Electrospray ionization ion mobility mass spectrometry (ESI‐IM‐MS) has been widely applied for structural characterization of apo‐state and ligand‐associated IDPs via two‐dimensional separation in the gas phase. Gas‐phase IDP structures have been regarded as kinetically trapped states originated from conformational features in solution. However, an implication of the states remains elusive in the structural characterization of IDPs, because it is unclear what structural property of IDPs is preserved. Recent studies have indicated that the conformational features of IDPs in solution are not fully reproduced in the gas phase. Nevertheless, the molecular interactions captured in the gas phase amplify the structural differences between IDP conformers. Therefore, an IDP conformational change that is not observed in solution is observable in the gas‐phase structures obtained by ESI‐IM‐MS. Herein, we have presented up‐to‐date researches on the key implications of kinetically trapped states in the gas phase with a brief summary of the structural dynamics of IDPs in ESI‐IM‐MS.

Fullerenes, cage-structured carbon allotropes, have been the subject of extensive research as new materials for diverse purposes. Yet, their formation process is still not clearly understood at the molecular level. In this study, we performed laser desorption ionization-ion mobility-mass spectrometry (LDI-IM-MS) of carbon substrates possessing different molecular sizes and structures to understand the formation process of fullerene. Our observations show that the formation process is strongly dependent on the size of the precursor used, with small precursors yielding small fullerenes and large graphitic precursors generally yielding larger fullerenes. These results clearly demonstrate that fullerene formation can proceed via both bottom-up and top-down processes, with the latter being favored for large precursors and more efficient at forming fullerenes. Furthermore, we observed that specific structures of carbon precursors could additionally affect the relative abundance of C60 fullerene. Overall, this study provides an advanced understanding of the mechanistic details underlying the formation processes of fullerene.