Native Ion Mobility-mass Spectrometry Research Articles

Mechanistic Insights into IscU Conformation Regulation for Fe-S Cluster Biogenesis Revealed by Variable Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry.

Iron-sulfur (Fe-S) cluster (ISC) cofactors are required for the function of many critical cellular processes. In the ISC Fe-S cluster biosynthetic pathway, IscU assembles Fe-S cluster intermediates from iron, electrons, and inorganic sulfur, which is provided by the cysteine desulfurase enzyme IscS. IscU also binds to Zn, which mimics and competes for binding with the Fe-S cluster. Crystallographic and nuclear magnetic resonance spectroscopic studies reveal that IscU is a metamorphic protein that exists in multiple conformational states, which include at least a structured form and a disordered form. The structured form of IscU is favored by metal binding and is stable in a narrow temperature range, undergoing both cold and hot denaturation. Interestingly, the form of IscU that binds IscS and functions in Fe-S cluster assembly remains controversial. Here, results from variable temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) establish that IscU exists in structured, intermediate, and disordered forms that rearrange to more extended conformations at higher temperatures. A comparison of Zn-IscU and apo-IscU reveals that Zn(II) binding attenuates the cold/heat denaturation of IscU, promotes refolding of IscU, favors the structured and intermediate conformations, and inhibits the disordered high charge states. Overall, these findings provide a structural rationalization for the role of Zn(II) in stabilizing IscU conformations and IscS in altering the IscU active site to prepare for Zn(II) release and cluster synthesis. This work highlights how vT-ESI-nIM-MS can be applied as a powerful tool in mechanistic enzymology by providing details of relationships among temperature, protein conformations, and ligand/protein binding.

Native Mass Spectrometry Coupled to Spectroscopic Methods to Investigate the Effect of Soybean Isoflavones on Structural Stability and Aggregation of Zinc Deficient and Metal-Free Superoxide Dismutase.

The deficiency or wrong combination of metal ions in Cu, Zn-superoxide dismutase (SOD1), is regarded as one of the main factors causing the aggregation of SOD1 and then inducing amyotrophic lateral sclerosis (ALS). A ligands-targets screening process based on native electrospray ionization ion mobility mass spectrometry (ESI-IMS-MS) was established in this study. Four glycosides including daidzin, sophoricoside, glycitin, and genistin were screened out from seven soybean isoflavone compounds and were found to interact with zinc-deficient or metal-free SOD1. The structure and conformation stability of metal-free and zinc-deficient SOD1 and their complexes with the four glycosides was investigated by collision-induced dissociation (CID) and collision-induced unfolding (CIU). The four glycosides could strongly bind to the metal-free and copper recombined SOD1 and enhance the folding stability of these proteins. Additionally, the ThT fluorescence assay showed that these glycosides could inhibit the toxic aggregation of the zinc-deficient or metal-free SOD1. The competitive interaction experiments together with molecular docking indicate that glycitin, which showed the best stabilizing effects, binds with SOD1 between β-sheet 6 and loop IV. In short, this study provides good insight into the relationship between inhibitors and different SOD1s.

Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase.

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Förster resonance energy transfer (tmFRET)-based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH2) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH2). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor-acceptor distance (rDA) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and rDA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.

Elucidating the Oligomerization and Cellular Interactions of a Trimer Derived from Aβ through Fluorescence and Mass Spectrometric Studies.

Aβ oligomers play a central role in the neurodegeneration observed with Alzheimer’s disease. Our laboratory has developed covalently stabilized trimers derived from residues 17–36 of Aβ as model systems for studying Aβ oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells and establish the occurrence of FRET, indicating the presence of oligomers within cells. Collectively, these multiple complementary techniques help better understand the complex behavior of the 2AT-L model system.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized β-sheet structures that accumulate in plaques in brains of Parkinson’s disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Investigation of Charge-State-Dependent Compaction of Protein Ions with Native Ion Mobility-Mass Spectrometry and Theory.

The precise relationship between native gas-phase protein ion structure, charge, desolvation, and activation remains elusive. Much evidence supports the Charge Residue Model for native protein ions formed by electrospray ionization, but scaling laws derived from it relate only to overall ion size. Closer examination of drift tube CCSs across individual native protein ion charge state distributions (CSDs) reveals deviations from global trends. To investigate whether this is due to structure variation across CSDs or contributions of long-range charge-dipole interactions, we performed in vacuo force field molecular dynamics (MD) simulations of multiple charge conformers of three proteins representing a variety of physical and structural features: β-lactoglobulin, concanavalin A, and glutamate dehydrogenase. Results from these simulated ions indicate subtle structure variation across their native CSDs, although effects of these structural differences and long-range charge-dependent interactions on CCS are small. The structure and CCS of smaller proteins may be more sensitive to charge due to their low surface-to-volume ratios and reduced capacity to compact. Secondary and higher order structure from condensed-phase structures is largely retained in these simulations, supporting the use of the term "native-like" to describe results from native ion mobility-mass spectrometry experiments, although, notably, the most compact structure can be the most different from the condensed-phase structure. Collapse of surface side chains to self-solvate through formation of new hydrogen bonds is a major feature of gas-phase compaction and likely occurs during the desolvation process. Results from these MD simulations provide new insight into the relationship of gas-phase protein ion structure, charge, and CCS.

Native Ion Mobility-Mass Spectrometry-Enabled Fast Structural Interrogation of Labile Protein Surface Modifications at the Intact Protein Level.

Protein sialylation has been closely linked to many diseases including Alzheimer's disease (AD). It is also broadly implicated in therapeutics operating in a pattern-dependent (e.g., Neu5Ac vs Neu5Gc) manner. However, how the sialylation pattern affects the AD-associated, transferrin-assisted iron/Aβ cellular uptake process remains largely ill-defined. Herein, we report the use of native ion mobility-mass spectrometry (IM-MS)-based fast structural probing methodology, enabling well-controlled, synergistic, and in situ manipulation of mature glycoproteins and attached sialic acids. IM-MS-centered experiments enable the combinatorial interrogation of sialylation effects on Aβ cytotoxicity and the chemical, conformational, and topological stabilities of transferrin. Cell viability experiments suggest that Neu5Gc replacement enhances the transferrin-assisted, iron loading-associated Aβ cytotoxicity. Native gel electrophoresis and IM-MS reveal that sialylation stabilizes transferrin conformation but inhibits its dimerization. Collectively, IM-MS is adapted to capture key sialylation intermediates involved in fine-tuning AD-associated glycoprotein structural microheterogeneity. Our results provide the molecular basis for the importance of sustaining moderate TF sialylation levels, especially Neu5Ac, in promoting iron cellular transportation and rescuing iron-enhanced Aβ cytotoxicity.

Bicarbonate buffers can promote crosslinking and alternative gas-phase dissociation pathways for multiprotein complexes

Previously, we have reported the stabilization effect of Hofmeister salts for multiprotein complexes (MPC) in the absence of bulk solvent (J. Am. Chem. Soc.2011,133 (29), 11,358–11367; Angew. Chem. Int. Ed.2012,51 (23), 5692–5695; Angew. Chem. Int. Ed.2013, 52 (32), 8329–8332.). Our efforts sought to bridge the gap between gas-phase protein structures and those found in solution. To reveal more detailed MPC topology information, native ion mobility-mass spectrometry (IM-MS) measurements are often combined with gas-phase activation methods. Conventional activation methods, including collision induced dissociation/unfolding (CID/CIU), however, primarily report information focused on monomeric subunits within the MPC, limiting the topological information obtained. Herein, we describe a simple buffer-doping method that promotes an alternative MPC CID pathway which readily produces product ions that correspond to larger sub-complexes from within some parent assemblies. Interestingly, tetramers exhibiting a dimer of dimers quaternary structure (e.g. hemoglobin and concanavalin A) produce dimeric product ions upon collisional activation following ionization from bicarbonate buffer, in contrast to the commonly observed monomer-ejection CID pathway. In order to both further investigate and validate our native IM-MS, we performed bottom-up proteomics experiments on MPCs housed in bicarbonate buffer. Our efforts revealed evidence of bicarbonate-mediated disulfide bond formation in proximal Cystine residues. We close by discussing the applications for these observations in the context of MPC structure determination by native IM-MS.

Ion mobility–mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy

Peripheral myelin protein (PMP22) is an integral membrane protein that traffics inefficiently even in wild-type (WT) form, with only 20% of the WT protein reaching its final plasma membrane destination in myelinating Schwann cells. Misfolding of PMP22 has been identified as a key factor in multiple peripheral neuropathies, including Charcot-Marie-Tooth disease and Dejerine-Sottas syndrome. While biophysical analyses of disease-associated PMP22 mutants show altered protein stabilities, leading to reduced surface trafficking and loss of PMP22 function, it remains unclear how destabilization of PMP22 mutations causes mistrafficking. Here, native ion mobility-mass spectrometry (IM-MS) is used to compare the gas phase stabilities and abundances for an array of mutant PM22 complexes. We find key differences in the PMP22 mutant stabilities and propensities to form homodimeric complexes. Of particular note, we observe that severely destabilized forms of PMP22 exhibit a higher propensity to dimerize than WT PMP22. Furthermore, we employ lipid raft-mimicking SCOR bicelles to study PMP22 mutants, and find that the differences in dimer abundances are amplified in this medium when compared to micelle-based data, with disease mutants exhibiting up to 4 times more dimer than WT when liberated from SCOR bicelles. We combine our findings with previous cellular data to propose that the formation of PMP22 dimers from destabilized monomers is a key element of PMP22 mistrafficking.

Interplay between chromophore binding and domain assembly by the B12-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B12-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B12 to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B12 photochemistry results in tetramer dissociation, releasing DNA for transcription. Although the details of the response of CarH to light are beginning to emerge, the biophysical mechanism of B12-binding in the dark and how this drives domain assembly is poorly understood. Here – using a combination of molecular dynamics simulations, native ion mobility mass spectrometry and time-resolved spectroscopy – we reveal a complex picture that varies depending on the availability of B12. When B12 is in excess, its binding drives structural changes in CarH monomers that result in the formation of head-to-tail dimers. The structural changes that accompany these steps mean that they are rate-limiting. The dimers then rapidly combine to form tetramers. Strikingly, when B12 is scarcer, as is likely in nature, tetramers with native-like structures can form without a B12 complement to each monomer, with only one apparently required per head-to-tail dimer. We thus show how a bulky chromophore such as B12 shapes protein/protein interactions and in turn function, and how a protein can adapt to a sub-optimal availability of resources. This nuanced picture should help guide the engineering of B12-dependent photoreceptors as light-activated tools for biomedical applications.

Structural modeling of a novel membrane-bound globin-coupled sensor in Geobacter sulfurreducens.

Globin-coupled sensors (GCS) usually consist of three domains: a sensor/globin, a linker, and a transmitter domain. The globin domain (GD), activated by ligand binding and/or redox change, induces an intramolecular signal transduction resulting in a response of the transmitter domain. Depending on the nature of the transmitter domain, GCSs can have different activities and functions, including adenylate and di-guanylate cyclase, histidine kinase activity, aerotaxis and/or oxygen sensing function. The gram-negative delta-proteobacterium Geobacter sulfurreducens expresses a protein with a GD covalently linked to a four transmembrane domain, classified, by sequence similarity, as GCS (GsGCS). While its GD is fully characterized, not so its transmembrane domain, which is rarely found in the globin superfamily. In the present work, GsGCS was characterized spectroscopically and by native ion mobility-mass spectrometry in combination with cryo-electron microscopy. Although lacking high resolution, the oligomeric state and the electron density map were valuable for further rational modeling of the full-length GsGCS structure. This model demonstrates that GsGCS forms a transmembrane domain-driven tetramer with minimal contact between the GDs and with the heme groups oriented outward. This organization makes an intramolecular signal transduction less likely. Our results, including the auto-oxidation rate and redox potential, suggest a potential role for GsGCS as redox sensor or in a membrane-bound e-/H+ transfer. As such, GsGCS might act as a player in connecting energy production to the oxidation of organic compounds and metal reduction. Database searches indicate that GDs linked to a four or seven helices transmembrane domain occur more frequently than expected.

Aptamer-ligand recognition studied by native ion mobility-mass spectrometry

The range of applications for aptamers, small oligonucleotide-based receptors binding to their targets with high specificity and affinity, has been steadily expanding. Our understanding of the mechanisms governing aptamer-ligand recognition and binding is however lagging, stymieing the progress in the rational design of new aptamers and optimization of the known ones. Here we demonstrate the capabilities and limitations of native ion mobility-mass spectrometry for the analysis of their higher-order structure and non-covalent interactions. A set of related cocaine-binding aptamers, displaying a range of folding properties and ligand binding affinities, was used as a case study in both positive and negative electrospray ionization modes. Using carefully controlled experimental conditions, we probed their conformational behavior and interactions with the high-affinity ligand quinine as a surrogate for cocaine. The ratios of bound and unbound aptamers in the mass spectra were used to rank them according to their apparent quinine-binding affinity, qualitatively matching the published ranking order. The arrival time differences between the free aptamer and aptamer-quinine complexes were consistent with a small ligand-induced conformational change, and found to inversely correlate with the affinity of binding. This mass spectrometry-based approach provides a fast and convenient way to study the molecular basis of aptamer-ligand recognition.

Enhanced Collision Induced Unfolding and Electron Capture Dissociation of Native-like Protein Ions.

Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.

Effects of Detergent on α-Synuclein Structure. A Native MS-Ion Mobility Study.

The intrinsically disordered protein α-synuclein plays a major role in Parkinson’s disease. The protein can oligomerize resulting in the formation of various aggregated species in neuronal cells, leading to neurodegeneration. The interaction of α-synuclein with biological cell membranes plays an important role for specific functions of α-synuclein monomers, e.g., in neurotransmitter release. Using different types of detergents to mimic lipid molecules present in biological membranes, including the presence of Ca2+ ions as an important structural factor, we aimed to gain an understanding of how α-synuclein interacts with membrane models and how this affects the protein conformation and potential oligomerization. We investigated detergent binding stoichiometry, affinity and conformational changes of α-synuclein taking detergent concentration, different detergent structures and charges into account. With native nano-electrospray ionization ion mobility-mass spectrometry, we were able to detect unique conformational patterns resulting from binding of specific detergents to α-synuclein. Our data demonstrate that α-synuclein monomers can interact with detergent molecules irrespective of their charge, that protein-micelle interactions occur and that micelle properties are an important factor.

Metal ions shape \u03b1-synuclein

α-Synuclein is an intrinsically disordered protein that can self-aggregate and plays a major role in Parkinson’s disease (PD). Elevated levels of certain metal ions are found in protein aggregates in neurons of people suffering from PD, and environmental exposure has also been linked with neurodegeneration. Importantly, cellular interactions with metal ions, particularly Ca2+, have recently been reported as key for α-synuclein’s physiological function at the pre-synapse. Here we study effects of metal ion interaction with α-synuclein at the molecular level, observing changes in the conformational behaviour of monomers, with a possible link to aggregation pathways and toxicity. Using native nano-electrospray ionisation ion mobility-mass spectrometry (nESI-IM-MS), we characterize the heterogeneous interactions of alkali, alkaline earth, transition and other metal ions and their global structural effects on α-synuclein. Different binding stoichiometries found upon titration with metal ions correlate with their specific binding affinity and capacity. Subtle conformational effects seen for singly charged metals differ profoundly from binding of multiply charged ions, often leading to overall compaction of the protein depending on the preferred binding sites. This study illustrates specific effects of metal coordination, and the associated electrostatic charge patterns, on the complex structural space of the intrinsically disordered protein α-synuclein.

Collision-Induced Unfolding Differentiates Functional Variants of the KCNQ1 Voltage Sensor Domain.

The KCNQ1 voltage-gated potassium channel regulates the repolarization of cardiac cells, and a plurality of point mutations in its voltage-sensing domain (VSD) are associated with toxic gain or loss of pore function, resulting in disease. As is the case with many disease-associated membrane proteins, there are hundreds of human variants of interest identified for KCNQ1; however, a significant portion of these variants have not been characterized in relation to their functional and disease associations. Additionally, as the VSD consists of four transmembrane helices, studies into dynamic structural differences among KCNQ1 VSD variants are hindered by the current limitations and deficits in the high-resolution structure determination of membrane proteins. Here, we use native ion mobility-mass spectrometry and collision-induced unfolding (CIU) to address the need for a high throughput-compatible method for the structural characterization of membrane protein variants of unknown significance using the KCNQ1 VSD as a model system. We perform CIU on wild-type and three mutant KCNQ1 VSD forms associated with the toxic gain or loss of function and show through both automated feature detection and comprehensive difference analysis of the CIU data sets that the variants are clearly grouped by function and disease association. We also construct a classification scheme based on the CIU data sets, which is able to differentiate the variant functional groups and classify a recently characterized variant to its correct grouping. Further, we probe the stability of the KCNQ1 VSD variants when liberated from C12E8 micelles at pH 8.0 and find preliminary evidence that the R231C mutation associated with the gain of the pore function is destabilized relative to the wild-type and loss of function variants.

Discovery of Potent Charge-Reducing Molecules for Native Ion Mobility Mass Spectrometry Studies.

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Rationally designed peptide-based inhibitor of Aβ42 fibril formation and toxicity: a potential therapeutic strategy for Alzheimer's disease.

Amyloid beta peptide (Aβ42) aggregation in the brain is thought to be responsible for the onset of Alzheimer's disease, an insidious condition without an effective treatment or cure. Hence, a strategy to prevent aggregation and subsequent toxicity is crucial. Bio-inspired peptide-based molecules are ideal candidates for the inhibition of Aβ42 aggregation, and are currently deemed to be a promising option for drug design. In this study, a hexapeptide containing a self-recognition component unique to Aβ42 was designed to mimic the β-strand hydrophobic core region of the Aβ peptide. The peptide is comprised exclusively of D-amino acids to enhance specificity towards Aβ42, in conjunction with a C-terminal disruption element to block the recruitment of Aβ42 monomers on to fibrils. The peptide was rationally designed to exploit the synergy between the recognition and disruption components, and incorporates features such as hydrophobicity, β-sheet propensity, and charge, that all play a critical role in the aggregation process. Fluorescence assays, native ion-mobility mass spectrometry (IM-MS) and cell viability assays were used to demonstrate that the peptide interacts with Aβ42 monomers and oligomers with high specificity, leading to almost complete inhibition of fibril formation, with essentially no cytotoxic effects. These data define the peptide-based inhibitor as a potentially potent anti-amyloid drug candidate for this hitherto incurable disease.