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We probe the adsorption of molecular H2O on a TiO2 (110)-(1 × 1) surface decorated with isolated VO clusters using ultrahigh-vacuum scanning tunneling microscopy (UHV-STM) and temperature-programmed desorption (TPD). Our STM images show that preadsorbed VO clusters on the TiO2 (110)-(1 × 1) surface induce the adsorption of H2O molecules at room temperature (RT). The adsorbed H2O molecules form strings of beads of H2O dimers bound to the 5-fold coordinated Ti atom (5c-Ti) rows and are anchored by VO. This RT adsorption is completely reversible and is unique to the VO-decorated TiO2 surface. TPD spectra reveal two new desorption states for VO stabilized H2O at 395 and 445 K, which is in sharp contrast to the desorption of water due to recombination of hydroxyl groups at 490 K from clean TiO2(110)-(1 × 1) surfaces. Density functional theory (DFT) calculations show that the binding energy of molecular H2O to the VO clusters on the TiO2 (110)-(1 × 1) surface is higher than binding to the bare surface by 0.42 eV, and the resulting H2O-VO-TiO2 (110) complex provides the anchor point for adsorption of the string of beads of H2O dimers.

Alzheimer's disease (AD) is rapidly reaching epidemic status among a burgeoning aging population. Much evidence suggests the toxicity of this amyloid disease is most influenced by the formation of soluble oligomeric forms of amyloid β-protein, particularly the 42-residue alloform (Aβ42). Developing potential therapeutics in a directed, streamlined approach to treating this disease is necessary. Here we utilize the joint pharmacophore space (JPS) model to design a new molecule [AC0107] incorporating structural characteristics of known Aβ inhibitors, blood-brain barrier permeability, and limited toxicity. To test the molecule's efficacy experimentally, we employed ion mobility mass spectrometry (IM-MS) to discover [AC0107] inhibits the formation of the toxic Aβ42 dodecamer at both high (1:10) and equimolar concentrations of inhibitor. Atomic force microscopy (AFM) experiments reveal that [AC0107] prevents further aggregation of Aβ42, destabilizes preformed fibrils, and reverses Aβ42 aggregation. This trend continues for long-term interaction times of 2 days until only small aggregates remain with virtually no fibrils or higher order oligomers surviving. Pairing JPS with IM-MS and AFM presents a powerful and effective first step for AD drug development. Graphical Abstract.

Amyloid formation is associated with devastating diseases such as Alzheimer's, Parkinson's and Type-2 diabetes. The large amyloid deposits found in patients suffering from these diseases have remained difficult to probe by structural means. Recent NMR models also predict heterotypic interactions from distinct peptide fragments but limited evidence of heterotypic packed sheets is observed in solution. Here we characterize two segments of the protein amyloid β (Aβ) known to form fibrils in Alzheimer's disease patients. We designed two variants of Aβ(19-24) and Aβ(27-32), IFAEDV (I6V) and NKGAIF (N6F) to lower the aggregation propensity of individual peptides while maintaining the similar interactions between the two segments in their native forms. We found that the variants do not form significant amyloid fibrils individually but a 1:1 mixture forms abundant fibrils. Using ion mobility-mass spectrometry (IM-MS), hetero-oligomers up to decamers were found in the mixture while the individual peptides formed primarily dimers and some tetramers consistent with a strong heterotypic interaction between the two segments. We showed by X-ray crystallography that I6V formed a Class 7 zipper with a weakly packed pair of β-sheets and no segregated dry interface, while N6F formed a more stable Class 1 zipper. In a mixture of equimolar N6F:I6V, I6V forms a more stable zipper than in I6V alone while no N6F or hetero-typic zippers are observed. These data are consistent with a mechanism where N6F catalyzes assembly of I6V into a stable zipper and perhaps into stable, pure I6V fibrils that are observed in AFM measurements.

The early oligomerization of amyloid β-protein (Aβ) is a crucial step in the etiology of Alzheimer's disease (AD), in which soluble and highly neurotoxic oligomers are produced and accumulated inside neurons. In search of therapeutic solutions for AD treatment and prevention, potent inhibitors that remodel Aβ assembly and prevent neurotoxic oligomer formation offer a promising approach. In particular, several polyphenolic compounds have shown anti-aggregation properties and good efficacy on inhibiting oligomeric amyloid formation. 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose is a large polyphenol that has been shown to be effective at inhibiting aggregation of full-length Aβ1-40 and Aβ1-42, but has the opposite effect on the C-terminal fragment Aβ25-35. Here, we use a combination of ion mobility coupled to mass spectrometry (IMS-MS), transmission electron microscopy (TEM) and molecular dynamics (MD) simulations to elucidate the inhibitory effect of PGG on aggregation of full-length Aβ1-40 and Aβ1-42. We show that PGG interacts strongly with these two peptides, especially in their N-terminal metal binding regions, and suppresses the formation of Aβ1-40 tetramer and Aβ1-42 dodecamer. By exploring multiple facets of polyphenol-amyloid interactions, we provide a molecular basis for the opposing effects of PGG on full-length Aβ and its C-terminal fragments.

Fibrils and oligomers are the aggregated protein agents of neuronal dysfunction in ALS diseases. Whereas we now know much about fibril architecture, atomic structures of disease-related oligomers have eluded determination. Here, we determine the corkscrew-like structure of a cytotoxic segment of superoxide dismutase 1 (SOD1) in its oligomeric state. Mutations that prevent formation of this structure eliminate cytotoxicity of the segment in isolation as well as cytotoxicity of the ALS-linked mutants of SOD1 in primary motor neurons and in a Danio rerio (zebrafish) model of ALS. Cytotoxicity assays suggest that toxicity is a property of soluble oligomers, and not large insoluble aggregates. Our work adds to evidence that the toxic oligomeric entities in protein aggregation diseases contain antiparallel, out-of-register β-sheet structures and identifies a target for structure-based therapeutics in ALS.

Ion mobility spectrometry-mass spectrometry (IMS-MS) methods are increasingly used to study noncovalent assemblies of peptides and proteins. This review focuses on the noncovalent self-assembly of amino acids and peptides, systems at the heart of the amyloid process that play a central role in a number of devastating diseases. Three different systems are discussed in detail: the 42-residue peptide amyloid-β42 implicated in the etiology of Alzheimer's disease, several amyloid-forming peptides with 6-11 residues, and the assembly of individual amino acids. We also discuss from a more fundamental perspective the processes that determine how quickly proteins and their assemblies denature when the analyte ion has been stripped of its solvent in an IMS-MS measurement and how to soften the measurement so that biologically meaningful data can be recorded.

We investigate the relationship between the inherent secondary structure and aggregation propensity of peptides containing chameleon sequences (i.e., sequences that can adopt either α or β structure depending on context) using a combination of replica exchange molecular dynamics simulations, ion-mobility mass spectrometry, circular dichroism, and transmission electron microscopy. We focus on an eight-residue long chameleon sequence that can adopt an α-helical structure in the context of the iron-binding protein from Bacillus anthracis (PDB id 1JIG ) and a β-strand in the context of the baculovirus P35 protein (PDB id 1P35 ). We show that the isolated chameleon sequence is intrinsically disordered, interconverting between α-helical and β-rich conformations. The inherent conformational plasticity of the sequence can be constrained by addition of flanking residues with a given secondary structure propensity. Intriguingly, we show that the chameleon sequence with helical flanking residues aggregates rapidly into fibrils, whereas the chameleon sequence with flanking residues that favor β-conformations has weak aggregation propensity. This work sheds new insights into the possible role of α-helical intermediates in fibril formation.

Despite extensive structure-function analyses, the molecular mechanisms of normal and pathological tau action remain poorly understood. How does the C-terminal microtubule-binding region regulate microtubule dynamics and bundling? In what biophysical form does tau transfer trans-synaptically from one neuron to another, promoting neurodegeneration and dementia? Previous biochemical/biophysical work led to the hypothesis that tau can dimerize via electrostatic interactions between two N-terminal 'projection domains' aligned in an anti-parallel fashion, generating a multivalent complex capable of interacting with multiple tubulin subunits. We sought to test this dimerization model directly. Native gel analyses of full-length tau and deletion constructs demonstrate that the N-terminal region leads to multiple bands, consistent with oligomerization. Ferguson analyses of native gels indicate that an N-terminal fragment (tau(45-230) ) assembles into heptamers/octamers. Ferguson analyses of denaturing gels demonstrates that tau(45-230) can dimerize even in sodium dodecyl sulfate. Atomic force microscopy reveals multiple levels of oligomerization by both full-length tau and tau(45-230) . Finally, ion mobility-mass spectrometric analyses of tau(106-144) , a small peptide containing the core of the hypothesized dimerization region, also demonstrate oligomerization. Thus, multiple independent strategies demonstrate that the N-terminal region of tau can mediate higher order oligomerization, which may have important implications for both normal and pathological tau action. The microtubule-associated protein tau is essential for neuronal development and maintenance, but is also central to Alzheimer's and related dementias. Unfortunately, the molecular mechanisms underlying normal and pathological tau action remain poorly understood. Here, we demonstrate that tau can homo-oligomerize, providing novel mechanistic models for normal tau action (promoting microtubule growth and bundling, suppressing microtubule shortening) and pathological tau action (poisoning of oligomeric complexes).

Targeting the early oligomerization of amyloid β protein (Aβ) is a promising therapeutic strategy for Alzheimer's disease (AD). Recently, certain C-terminal fragments (CTFs) derived from Aβ42 were shown to be potent inhibitors of Aβ-induced toxicity. The shortest peptide studied, Aβ(39-42), has been shown to modulate Aβ oligomerization and inhibit Aβ toxicity. Understanding the mechanism of these CTFs, especially Aβ(39-42), is of significance for future therapeutic development of AD and peptidomimetic-based drug development. Here we used ion mobility spectrometry-mass spectrometry to investigate the interactions between two modified Aβ(39-42) derivatives, VVIA-NH2 and Ac-VVIA, and full-length Aβ42. VVIA-NH2 was previously shown to inhibit Aβ toxicity, whereas Ac-VVIA did not. Our mass spectrometry analysis revealed that VVIA-NH2 binds directly to Aβ42 monomer and small oligomers while Ac-VVIA binds only to Aβ42 monomer. Ion mobility studies showed that VVIA-NH2 modulates Aβ42 oligomerization by not only inhibiting the dodecamer formation but also disaggregating preformed Aβ42 dodecamer. Ac-VVIA also inhibits and removes preformed Aβ42 dodecamer. However, the Aβ42 sample with the addition of Ac-VVIA clogged the nanospray tip easily, indicating that larger aggregates are formed in the solution in the presence of Ac-VVIA. Molecular dynamics simulations suggested that VVIA-NH2 binds specifically to the C-terminal region of Aβ42 while Ac-VVIA binds dispersedly to multiple regions of Aβ42. This work implies that C-terminal interactions and binding to Aβ oligomers are important for C-terminal fragment inhibitors.

Evidence suggests that oligomers of the 42-residue form of the amyloid β-protein (Aβ), Aβ42, play a critical role in the etiology of Alzheimer's disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aβ42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aβ42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 μM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar β-sheet structures. The preprotofibrils are a single Aβ42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aβ40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimer's disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

A variety of species express the amyloid β-protein (Aβ (the term "Aβ" refers both to Aβ40 and Aβ42, whereas "Aβ40" and "Aβ42" refer to each isoform specifically). Those species expressing Aβ with primary structure identical to that expressed in humans have been found to develop amyloid deposits and Alzheimer's disease-like neuropathology. In contrast, the Aβ sequence in mice and rats contains three amino acid substitutions, Arg5Gly, His13Arg, and Tyr10Phe, which apparently prevent the development of AD-like neuropathology. Interestingly, the brush-tailed rat, Octodon degus, expresses Aβ containing only one of these substitutions, His13Arg, and does develop AD-like pathology. We investigate here the biophysical and biological properties of Aβ peptides from humans, mice (Mus musculus), and rats (Octodon degus). We find that each peptide displays statistical coil → β-sheet secondary structure transitions, transitory formation of hydrophobic surfaces, oligomerization, formation of annuli, protofibrils, and fibrils, and an inverse correlation between rate of aggregation and aggregate size (faster aggregation produced smaller aggregates). The rank order of assembly rate was mouse > rat > Aβ42. The rank order of neurotoxicity of assemblies formed by each peptide immediately after preparation was Aβ42 > mouse ≈ rat. These data do not support long-standing hypotheses that the primary factor controlling development of AD-like neuropathology in rodents is Aβ sequence. Instead, the data support a hypothesis that assembly quaternary structure and organismal responses to toxic peptide assemblies mediate neuropathogenetic effects. The implication of this hypothesis is that a valid understanding of disease causation within a given system (organism, tissue, etc.) requires the coevaluation of both biophysical and cell biological properties of that system.

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, (273)GKVQIINKKLDL(284) (here "R2/WT"), was modified with a spin-label at the N-terminus. With the resulting peptide, R2/G273C-SL, we probed events at time scales spanning seconds to hours after aggregation is initiated using transmission electron microscopy (TEM), thioflavin T (THT) fluorescence, ion mobility mass spectrometry (IMMS), electron paramagnetic resonance (EPR), and Overhauser dynamic nuclear polarization (ODNP) to determine if deliberate changes to its conformational states and population in solution influence downstream propensity to form fibrillar aggregates. We find varying solution conditions by adding the osmolyte urea or TMAO, or simply using different buffers (acetate buffer, phosphate buffer, or water), produces significant differences in early monomer/dimer populations and conformations. Crucially, these characteristics of the peptide in solution state before aggregation is initiated dictate the fibril formation propensity after aggregation. We conclude the driving forces that accelerate aggregation, when heparin is added, do not override the subtle intra- or interprotein interactions induced by the initial solvent conditions. In other words, the balance of protein-protein vs protein-solvent interactions present in the initial solution conditions is a critical driving force for fibril formation.

Oligomeric states of the amyloid β-protein (Aβ) appear to be causally related to Alzheimer's disease (AD). Recently, two familial mutations in the amyloid precursor protein gene have been described, both resulting in amino acid substitutions at Ala2 (A2) within Aβ. An A2V mutation causes autosomal recessive early onset AD. Interestingly, heterozygotes enjoy some protection against development of the disease. An A2T substitution protects against AD and age-related cognitive decline in non-AD patients. Here, we use ion mobility-mass spectrometry (IM-MS) to examine the effects of these mutations on Aβ assembly. These studies reveal different assembly pathways for early oligomer formation for each peptide. A2T Aβ42 formed dimers, tetramers, and hexamers, but dodecamer formation was inhibited. In contrast, no significant effects on Aβ40 assembly were observed. A2V Aβ42 also formed dimers, tetramers, and hexamers, but it did not form dodecamers. However, A2V Aβ42 formed trimers, unlike A2T or wild-type (wt) Aβ42. In addition, the A2V substitution caused Aβ40 to oligomerize similar to that of wt Aβ42, as evidenced by the formation of dimers, tetramers, hexamers, and dodecamers. In contrast, wt Aβ40 formed only dimers and tetramers. These results provide a basis for understanding how these two mutations lead to, or protect against, AD. They also suggest that the Aβ N-terminus, in addition to the oft discussed central hydrophobic cluster and C-terminus, can play a key role in controlling disease susceptibility.

Aggregation of proteins to fiberlike aggregates often involves a transformation of native monomers to β-sheet-rich oligomers. This general observation underestimates the importance of α-helical segments in the aggregation cascade. Here, using a combination of experimental techniques and accelerated molecular dynamics simulations, we investigate the aggregation of a 43-residue, apolipoprotein A-I mimetic peptide and its E21Q and D26N mutants. Our study indicates a strong propensity of helical segments not to adopt cross-β-fibrils. The helix-turn-helix monomeric conformation of the peptides is preserved in the mature fibrils. Furthermore, we reveal opposite effects of mutations on and near the turn region in the self-assembly of these peptides. We show that the E21-R24 salt bridge is a major contributor to helix-turn-helix folding, subsequently leading to abundant fibril formation. On the other hand, the K19-D26 interaction is not required to fold the native helix-turn-helix peptide. However, removal of the charged D26 residue decreases the stability of the helix-turn-helix monomer and consequently reduces the level of aggregation. Finally, we provide a more refined assembly model for the helix-turn-helix peptides from apolipoprotein A-I based on the parallel stacking of helix-turn-helix dimers.

The early oligomerization of amyloid β-protein (Aβ) has been shown to be an important event in the pathology of Alzheimer's disease (AD). Designing small molecule inhibitors targeting Aβ oligomerization is one attractive and promising strategy for AD treatment. Here we used ion mobility spectrometry coupled to mass spectrometry (IMS-MS) to study the different effects of the molecular tweezers CLR01 and CLR03 on Aβ self-assembly. CLR01 was found to bind to Aβ directly and disrupt its early oligomerization. Moreover, CLR01 remodeled the early oligomerization of Aβ42 by compacting the structures of dimers and tetramers and as a consequence eliminated higher-order oligomers. Unexpectedly, the negative-control derivative, CLR03, which lacks the hydrophobic arms of the tweezer structure, was found to facilitate early Aβ oligomerization. Our study provides an example of IMS as a powerful tool to study and better understand the interaction between small molecule modulators and Aβ oligomerization, which is not attainable by other methods, and provides important insights into therapeutic development of molecular tweezers for AD treatment.

Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; (273)GKVQIINKKLDL(284)) and PHF6 (R3/wt; (306)VQIVYKPVDLSK(317)) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*-PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function.

We have investigated at the oligomeric level interactions between Aβ(25-35) and Tau(273-284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer's disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273-284) shows a high affinity to form heteroligomers with existing Aβ(25-35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25-35) or Tau(273-284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25-35) fibrils. The incorporation of some Tau into β-rich Aβ(25-35) oligomers reduces the aggregation propensity of Aβ(25-35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25-35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

We report the results of a systematic study of the catalytic activity of mass-selected vanadium oxide clusters deposited on rutile TiO2 surfaces under ultrahigh vacuum (UHV) conditions. Our results show that supported V, VO, and VO2 clusters are not catalytically active for the oxidative dehydrogenation of methanol to formaldehyde but can be made catalytically active by postoxidation. In addition, we found that the postoxidized VO/TiO2 produces the most formaldehyde. Scanning tunneling microscopy (STM) imaging of the postoxidized VO/TiO2 reveals isolated clusters with height and width indicative of VO3 bound to the TiO2 surface. Our results are consistent with previous density functional theory (DFT) calculations that predict that VO3 will be produced by postoxidation of VO and that VO3/TiO2 is an active catalyst.

Human islet amyloid polypeptide (hIAPP or Amylin) is a 37 residue hormone that is cosecreted with insulin from the pancreatic islets. The aggregation of hIAPP plays a role in the progression of type 2 diabetes and contributes to the failure of islet cell grafts. Despite considerable effort, little is known about the mode of action of IAPP amyloid inhibitors, and this has limited rational drug design. Insulin is one of the most potent inhibitors of hIAPP fibril formation, but its inhibition mechanism is not understood. In this study, the aggregation of mixtures of hIAPP with insulin, as well as with the separate A and B chains of insulin, were characterized using ion mobility spectrometry-based mass spectrometry and atomic force microscopy. Insulin and the insulin B chain target the hIAPP monomer in its compact isoform and shift the equilibrium away from its extended isoform, an aggregation-prone conformation, and thus inhibit hIAPP from forming β-sheets and subsequently amyloid fibrils. All-atom molecular modeling supports these conclusions.

We have investigated at the oligomeric level interactions between Aβ(25-35) and Tau(273-284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer's disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273-284) shows a high affinity to form heteroligomers with existing Aβ(25-35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25-35) or Tau(273-284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25-35) fibrils. The incorporation of some Tau into β-rich Aβ(25-35) oligomers reduces the aggregation propensity of Aβ(25-35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25-35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

Ion mobility is not a newly discovered phenomenon. It has roots going back to Langevin at the beginning of the 20th century. Our group initially got involved by accident around 1990 and this paper is a brief account of what has transpired here at UCSB the past 25 years in response to this happy accident. We started small, literally, with transition metal atomic ions and transitioned to carbon clusters, synthetic polymers, most types of biological molecules and eventually peptide and protein oligomeric assembly. Along the way we designed and built several generations of instruments, a process that is still ongoing. And perhaps most importantly we have incorporated theory with experiment from the beginning; a necessary wedding that allows an atomistic face to be put on the otherwise interesting but not fully informative cross section measurements.

Five different mutants of [Leu-5] Enkephalin YGGFL peptide have been investigated for fibril formation propensities. The early oligomer structures have been probed with a combination of ion-mobility mass spectrometry and computational modeling. The two peptides YVIFL and YVVFL form oligomers and amyloid-like fibrils. YVVFV shows an early stage oligomer distribution similar to those of the previous two, but amyloid-like aggregates are less abundant. Atomic resolution X-ray structures of YVVFV show two different modes of interactions at the dry interface between steric zippers and pairs of antiparallel β-sheets, but both are less favorable than the packing motif found in YVVFL. Both YVVFV and YVVFL can form a Class 6 steric zipper. However, in YVVFV, the strands between mating sheets are parallel to each other and in YVVFL they are antiparallel. The overall data highlight the importance of structurally characterizing high order oligomers within oligomerization pathways in studies of nanostructure assembly.

Five different mutants of [Leu-5] Enkephalin YGGFL peptide have been investigated for fibril formation propensities. The early oligomer structures have been probed with a combination of ion-mobility mass spectrometry and computational modeling. The two peptides YVIFL and YVVFL form oligomers and amyloid-like fibrils. YVVFV shows an early stage oligomer distribution similar to those of the previous two, but amyloid-like aggregates are less abundant. Atomic resolution X-ray structures of YVVFV show two different modes of interactions at the dry interface between steric zippers and pairs of antiparallel β-sheets, but both are less favorable than the packing motif found in YVVFL. Both YVVFV and YVVFL can form a Class 6 steric zipper. However, in YVVFV, the strands between mating sheets are parallel to each other and in YVVFL they are antiparallel. The overall data highlight the importance of structurally characterizing high order oligomers within oligomerization pathways in studies of nanostructure assembly.

Alzheimer's disease (AD) is characterized by multiple, intertwined pathological features, including amyloid-β (Aβ) aggregation, metal ion dyshomeostasis, and oxidative stress. We report a novel compound (ML) prototype of a rationally designed molecule obtained by integrating structural elements for Aβ aggregation control, metal chelation, reactive oxygen species (ROS) regulation, and antioxidant activity within a single molecule. Chemical, biochemical, ion mobility mass spectrometric, and NMR studies indicate that the compound ML targets metal-free and metal-bound Aβ (metal-Aβ) species, suppresses Aβ aggregation in vitro, and diminishes toxicity induced by Aβ and metal-treated Aβ in living cells. Comparison of ML to its structural moieties (i.e., 4-(dimethylamino)phenol (DAP) and (8-aminoquinolin-2-yl)methanol (1)) for reactivity with Aβ and metal-Aβ suggests the synergy of incorporating structural components for both metal chelation and Aβ interaction. Moreover, ML is water-soluble and potentially brain permeable, as well as regulates the formation and presence of free radicals. Overall, we demonstrate that a rational structure-based design strategy can generate a small molecule that can target and modulate multiple factors, providing a new tool to uncover and address AD complexity.

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single β-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early β-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms.

Residue mutations have substantial effects on aggregation kinetics and propensities of amyloid peptides and their aggregate morphologies. Such effects are attributed to conformational transitions accessed by various types of oligomers such as steric zipper or single β-sheet. We have studied the aggregation propensities of six NNQQNY mutants: NVVVVY, NNVVNV, NNVVNY, VIQVVY, NVVQIY, and NVQVVY in water using a combination of ion-mobility mass spectrometry, transmission electron microscopy, atomic force microscopy, and all-atom molecular dynamics simulations. Our data show a strong correlation between the tendency to form early β-sheet oligomers and the subsequent aggregation propensity. Our molecular dynamics simulations indicate that the stability of a steric zipper structure can enhance the propensity for fibril formation. Such stability can be attained by either hydrophobic interactions in the mutant peptide or polar side-chain interdigitations in the wild-type peptide. The overall results display only modest agreement with the aggregation propensity prediction methods such as PASTA, Zyggregator, and RosettaProfile, suggesting the need for better parametrization and model peptides for these algorithms.

Although most cases of Alzheimer's disease (AD) are sporadic, ∼5% of cases are genetic in origin. These cases, known as familial Alzheimer's disease (FAD), are caused by mutations that alter the rate of production or the primary structure of the amyloid β-protein (Aβ). Changes in the primary structure of Aβ alter the peptide's assembly and toxic activity. Recently, a primary working hypothesis for AD has evolved where causation has been attributed to early, soluble peptide oligomer states. Here we posit that both experimental and pathological differences between FAD-related mutants and wild-type Aβ could be reflected in the early oligomer distributions of these peptides. We use ion mobility-based mass spectrometry to probe the structure and early aggregation states of three mutant forms of Aβ40 and Aβ42: Tottori (D7N), Flemish (A21G), and Arctic (E22G). Our results indicate that the FAD-related amino acid substitutions have no noticeable effect on Aβ monomer cross section, indicating there are no major structural changes in the monomers. However, we observe significant changes to the aggregation states populated by the various Aβ mutants, indicating that structural changes present in the monomers are reflected in the oligomers. Moreover, the early oligomer distributions differ for each mutant, suggesting a possible structural basis for the varied pathogenesis of different forms of FAD.

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinson's disease and Alzheimer's disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimer's disease.

Abeta40 and Abeta42 are peptides that adopt similar random-coil structures in solution. Abeta42, however, is significantly more neurotoxic than Abeta40 and forms amyloid fibrils much more rapidly than Abeta40. Here, mass spectrometry and ion mobility spectrometry are used to investigate a mixture of Abeta40 and Abeta42. The mass spectrum for the mixed solution shows the presence of a heterooligomer composed of equal parts of Abeta40 and Abeta42. Ion mobility results indicate that this mixed species comprises an oligomer distribution extending to tetramers. Abeta40 alone produces such a distribution, whereas Abeta42 alone produces oligomers as large as dodecamers. This indicates that Abeta40 inhibits Abeta42 oligomerization.

Abeta40 and Abeta42 are peptides that adopt similar random-coil structures in solution. Abeta42, however, is significantly more neurotoxic than Abeta40 and forms amyloid fibrils much more rapidly than Abeta40. Here, mass spectrometry and ion mobility spectrometry are used to investigate a mixture of Abeta40 and Abeta42. The mass spectrum for the mixed solution shows the presence of a heterooligomer composed of equal parts of Abeta40 and Abeta42. Ion mobility results indicate that this mixed species comprises an oligomer distribution extending to tetramers. Abeta40 alone produces such a distribution, whereas Abeta42 alone produces oligomers as large as dodecamers. This indicates that Abeta40 inhibits Abeta42 oligomerization.

The structure of the 21-30 fragment of the amyloid beta-protein (Abeta) was investigated by ion mobility mass spectrometry and replica exchange dynamics simulations. Mutations associated with familial Alzheimer's disease (E22G, E22Q, E22K, and D23N) of Abeta(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the WT peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding. Results for the mutants show that substitutions at E22 do little to alter the overall structure of the fragment. A substitution at D23 resulted in a change of structure for Abeta(21-30). A comparison of these gas-phase studies to previous solution-phase studies reveals that the peptide can fold in the absence of solvent to a structure also seen in solution, highlighting the important role of the D23 hydrogen bonding network in stabilizing the fragment's folded structure.

The structure of the 21-30 fragment of the amyloid beta-protein (Abeta) was investigated by ion mobility mass spectrometry and replica exchange dynamics simulations. Mutations associated with familial Alzheimer's disease (E22G, E22Q, E22K, and D23N) of Abeta(21-30) were also studied, in order to understand any structural changes that might occur with these substitutions. The structure of the WT peptide shows a bend and a perpendicular turn in the backbone which is maintained by a network of D23 hydrogen bonding. Results for the mutants show that substitutions at E22 do little to alter the overall structure of the fragment. A substitution at D23 resulted in a change of structure for Abeta(21-30). A comparison of these gas-phase studies to previous solution-phase studies reveals that the peptide can fold in the absence of solvent to a structure also seen in solution, highlighting the important role of the D23 hydrogen bonding network in stabilizing the fragment's folded structure.

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid beta-protein (Abeta) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Abeta. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Abeta. Enhanced aggregation in Abeta with familial Alzheimer's disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Abeta. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Abeta folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Abeta are fundamentally different.

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimer's disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid beta-protein (Abeta) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Abeta. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Abeta. Enhanced aggregation in Abeta with familial Alzheimer's disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Abeta. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Abeta folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Abeta are fundamentally different.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.

Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer's and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid beta-protein, the key pathogenetic agent in Alzheimer's disease.

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.