Amino Acid Metaclusters: Implications of Growth Trends on Peptide Self-Assembly and Structure

Ion-mobility mass spectrometry is utilized to examine the metacluster formation of serine, asparagine, isoleucine, and tryptophan. These amino acids are representative of different classes of noncharged amino acids. We show that they can form relatively large metaclusters in solution that are difficult or impossible to observe by traditional solution techniques. We further demonstrate, as an example, that the formation of Ser metaclusters is not an ESI artifact because large metaclusters can be detected in negative polarity and low concentration with similar cross sections to those measured in positive polarity and higher concentration. The growth trends of tryptophan and isoleucine metaclusters, along with serine, asparagine, and the previously studied phenylalanine, are balanced among various intrinsic properties of individual amino acids (e.g., hydrophobicity, size, and shape). The metacluster cross sections of hydrophilic residues (Ser, Asn, Trp) tend to stay on or fall below the isotropic model trend lines whereas those of hydrophobic amino acids (Ile, Phe) deviate positively from the isotropic trend lines. The growth trends correlate well to the predicted aggregation propensity of individual amino acids. From the metacluster data, we introduce a novel approach to score and predict aggregation propensity of peptides, which can offer a significant improvement over the existing methods in terms of accuracy. Using a set of hexapeptides, we show that the strong negative deviations of Ser metaclusters from the isotropic model leads a prediction of microcrystalline formation for the SFSFSF peptide, whereas the strong positive deviation of Ile leads to prediction or fibril formation for the NININI peptide. Both predictions are confirmed experimentally using ion mobility and TEM measurements. The peptide SISISI is predicted to only weakly aggregate, a prediction confirmed by TEM

Tau Aggregation Propensity Engrained in Its Solution State

A peptide fragment of the human tau protein which stacks to form neat cross β-sheet fibrils, resembling that found in pathological aggregation, ²⁷³GKVQ­IINK­KLDL²⁸⁴ (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 <i>before</i> aggregation is initiated dictate the fibril formation propensity <i>after</i> 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

Opposing Effects of Cucurbit[7]uril and 1,2,3,4,6-Penta‑<i>O</i>‑galloyl-β‑d‑glucopyranose on Amyloid β_25–35 Assembly

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by extracellular deposits of amyloid β protein (Aβ) in the brain. The conversion of soluble monomers to amyloid Aβ fibrils is a complicated process and involves several transient oligomeric species, which are widely believed to be highly toxic and play a crucial role in the etiology of AD. The development of inhibitors to prevent formation of small and midsized oligomers is a promising strategy for AD treatment. In this work, we employ ion mobility spectrometry (IMS), transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to elucidate the structural modulation promoted by two potential inhibitors of Aβ oligomerization, cucurbit[7]­uril (CB[7]) and 1,2,3,4,6-penta-<i>O</i>-galloyl-β-d-glucopyranose (PGG), on early oligomer and fibril formation of the Aβ_25–35 fragment. One and two CB[7] molecules bind to Aβ_25–35 monomers and dimers, respectively, and suppress aggregation by remodeling early oligomer structures and inhibiting the formation of higher-order oligomers. On the other hand, nonselective binding was observed between PGG and Aβ_25–35. The interactions between PGG and Aβ_25–35, surprisingly, enhanced the formation of Aβ aggregates by promoting extended Aβ_25–35 conformations in both homo- and hetero-oligomers. When both ligands were present, the inhibitory effect of CB[7] overrode the stimulatory effect of PGG on Aβ_25–35 aggregation, suppressing the formation of large amyloid oligomers and eliminating the structural conversion from isotropic to β-rich topologies induced by PGG. Our results provide mechanistic insights into CB[7] and PGG action on Aβ oligomerization. They also demonstrate the power of the IMS technique to investigate mechanisms of multiple small-molecule agents on the amyloid formation process

Effects of pH and Charge State on Peptide Assembly: The YVIFL Model System

Peptide oligomerization is necessary but not sufficient for amyloid fibril formation. Here, we use a combination of experiments and simulations to understand how pH influences the aggregation properties of a small hydrophobic peptide, YVIFL, which is a mutant form of [Leu-5]-Enkephalin. Transmission electron microscopy and atomic force microscopy measurements reveal that this peptide forms small aggregates under acidic conditions (pH = 2), but that extensive fibrillization only occurs under basic conditions (pH = 9 and 11). Ion-mobility mass spectrometry identifies key oligomers in the oligomerization process, which are further characterized at an atomistic level by molecular dynamics simulations. These simulations suggest that terminal charges play a critical role in determining aggregation propensity and aggregate morphology. They also reveal the presence of steric zipper oligomers under basic conditions, a possible precursor to fibril formation. Our experiments suggest that multiple aggregation pathways can lead to YVIFL fibrils, and that cooperative and multibody interactions are key mechanistic elements in the early stages of aggregation

Schematic map of the six CNS tau isoforms.

<p>Exons 2 (E2), 3 (E3) and 10 (E10) are alternatively spliced to generate all six possible combinations. Arrowheads denote the position of the G55R mutation, present in four of the six isoforms. R1, R2, R3 and R4 denote the four imperfect repeats in the MT binding region.</p

A. The family tree of the affected family shows the pattern of inheritance.

<p>The proband is the black oval on the left side of the figure (II:1), marked with an arrow. Tau haplotypes of sequenced individuals are also noted. “aoo” corresponds to age of onset; “aod” corresponds to age of death; black filling indicates persons possessing the G55R mutation; gray filling corresponds to diagnosed dementia of unknown origin (presumed to be G55R but inadequate medical records exist). Proband's son III:1 (from first marriage) is 36 years old and a carrier of G55R. Proband's second son III:2 (from second marriage) is 31 and also a G55R carrier. The other two sons (III:3 and III:4; from the second marriage) are not G55R carriers and are 29 and 28 years old. <b>B. The tau sequence in the region of the G55R mutation is extremely highly conserved across species lines.</b> The glycine at position 55 is completely conserved in seven species ranging from humans to lizards. Color coding emphasizes conserved nature of acidic (red), basic (blue), hydrophilic/polar (orange), hydrophobic (green) and proline (peach) positions.</p

The G55R mutation increases the ability of 4R tau but not 3R tau to nucleate microtubule assembly.

<p>(A) Microtubule assembly in reactions containing a 1∶30 tau:tubulin dimer molar ratio were assayed by light scattering as a function of time. (B) Co-sedimentation assays demonstrate that the G55R mutation does not affect the ability of tau to assemble MT mass at steady-state, nor does it affect the ability of tau to bind to microtubules. Statistical significance was determined by comparing each mutant to its corresponding WT using two-tailed t-tests. Data in both panels represent the mean ± SEM from three independent experiments.</p

Tau Assembly: The Dominant Role of PHF6 (VQIVYK) in Microtubule Binding Region Repeat R3

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; ²⁷³GK­<u>VQIINK</u>­KLDL²⁸⁴) and PHF6 (R3/wt; ³⁰⁶<u>VQIVYK</u>­PVDLSK³¹⁷) 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

Factors That Drive Peptide Assembly and Fibril Formation: Experimental and Theoretical Analysis of Sup35 NNQQNY Mutants

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

Amyloid β‑Protein C‑Terminal Fragments: Formation of Cylindrins and β‑Barrels

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