Defining a Physical Basis for Diversity in Protein Self-Assemblies Using a Minimal Model

Self-assembly of proteins into ordered, fibrillar structures is a commonly observed theme in biology. It has been observed that diverse set of proteins (e.g., alpha-synuclein, insulin, TATA-box binding protein, Sup35, p53), independent of their sequence, native structure, or function could self-assemble into highly ordered structures known as amyloids. What are the crucial features underlying amyloidogenesis that make it so generic? Using coarse-grained simulations of peptide self-assembly, we argue that variation in two physical parametersbending stiffness of the polypeptide and strength of intermolecular interactionscan give rise to many of the structural features typically associated with amyloid self-assembly. We show that the interplay between these two factors gives rise to a rich phase diagram displaying high diversity in aggregated states. For certain parameters, we find a bimodal distribution for the order parameter implying the coexistence of ordered and disordered aggregates. Our findings may explain the experimentally observed variability including the “off-pathway” aggregated structures. Further, we demonstrate that sequence-dependence and protein-specific signatures could be mapped to our coarse-grained framework to study self-assembly behavior of realistic systems such as the STVIIE peptide and Aβ42. The work also provides certain guiding principles that could be used to design novel peptides with desired self-assembly properties, by tuning a few physical parameters

Defining a Physical Basis for Diversity in Protein Self-Assemblies Using a Minimal Model

Self-assembly of proteins into ordered, fibrillar structures is a commonly observed theme in biology. It has been observed that diverse set of proteins (e.g., alpha-synuclein, insulin, TATA-box binding protein, Sup35, p53), independent of their sequence, native structure, or function could self-assemble into highly ordered structures known as amyloids. What are the crucial features underlying amyloidogenesis that make it so generic? Using coarse-grained simulations of peptide self-assembly, we argue that variation in two physical parametersbending stiffness of the polypeptide and strength of intermolecular interactionscan give rise to many of the structural features typically associated with amyloid self-assembly. We show that the interplay between these two factors gives rise to a rich phase diagram displaying high diversity in aggregated states. For certain parameters, we find a bimodal distribution for the order parameter implying the coexistence of ordered and disordered aggregates. Our findings may explain the experimentally observed variability including the “off-pathway” aggregated structures. Further, we demonstrate that sequence-dependence and protein-specific signatures could be mapped to our coarse-grained framework to study self-assembly behavior of realistic systems such as the STVIIE peptide and Aβ42. The work also provides certain guiding principles that could be used to design novel peptides with desired self-assembly properties, by tuning a few physical parameters

Site-Specific Fluorescence Dynamics of α‑Synuclein Fibrils Using Time-Resolved Fluorescence Studies: Effect of Familial Parkinson’s Disease-Associated Mutations

α-Synuclein (α-Syn) aggregation is directly implicated in both the initiation and spreading of Parkinson’s Diseases (PD) pathogenesis. Although the familial PD-associated mutations (A53T, E46K, and A30P) are known to affect the aggregation kinetics of α-Syn <i>in vitro</i>, their structural differences in resultant fibrils are largely unknown. In this report we studied the site-specific dynamics of wild type (wt) α-Syn and its three PD mutant fibrils using time-resolved fluorescence intensity, anisotropy decay kinetics, and fluorescence quenching. Our data suggest that the N- and C-terminus are more flexible and exposed compared to the middle non-amyloid-β component (NAC) region of wt and PD mutant α-Syn fibrils. Yet the N-terminus showed great conformational heterogeneity compared to the C-terminus for all these proteins. 71 position of E46K showed more flexibility and solvent exposure compared to other α-Syns, whereas both E46K and A53T fibrils possess a more rigid C-terminus compared to wt and A30P. The present data suggest that wt and PD mutant fibrils possess large differences in flexibility and solvent exposure at different positions, which may contribute to their different pathogenicity in PD

Snapshots at various time steps of ACTH-β-end simulation system showing hetero-oligomerization.

<p>The hetero-trimer consisting of two β-end and one ACTH is stable up to end of 20 ns simulation. β-end A, β-end B, ACTH C and ACTH D are represented by blue, yellow, green and purple color, respectively.</p

Analysis of the interpeptide interactions in ACTH-β-end system.

<p>A) Contact map showing various residues in contact within the ACTH-β-end simulation box at t = 20 ns. B) Schematic representation of the stable hydrogen bonds observed between the peptides during the simulation.</p

Curcumin Modulates α‑Synuclein Aggregation and Toxicity

In human beings, Parkinson’s disease (PD) is associated with the oligomerization and amyloid formation of α-synuclein (α-Syn). The polyphenolic Asian food ingredient curcumin has proven to be effective against a wide range of human diseases including cancers and neurological disorders. While curcumin has been shown to significantly reduce cell toxicity of α-Syn aggregates, its mechanism of action remains unexplored. Here, using a series of biophysical techniques, we demonstrate that curcumin reduces toxicity by binding to preformed oligomers and fibrils and altering their hydrophobic surface exposure. Further, our fluorescence and two-dimensional nuclear magnetic resonance (2D-NMR) data indicate that curcumin does not bind to monomeric α-Syn but binds specifically to oligomeric intermediates. The degree of curcumin binding correlates with the extent of α-Syn oligomerization, suggesting that the ordered structure of protein is required for effective curcumin binding. The acceleration of aggregation by curcumin may decrease the population of toxic oligomeric intermediates of α-Syn. Collectively; our results suggest that curcumin and related polyphenolic compounds can be pursued as candidate drug targets for treatment of PD and other neurological diseases

MD simulation of ACTH-β-end system.

<p>All-atom MD simulation for the duration of 20 ns was performed in explicit solvent using two ACTH and two β-end. <b>A</b>) Snapshots at the left-hand side indicating the initial configuration (t = 0 ns) and at right-hand side indicating the final configuration (t = 20 ns). <b>B</b>) Plot of distances between center of masses (Dij) of peptides against time indicating close proximity of peptides A, B and D. <b>C</b>) Time progression of contact regions between β-end A and β-end B. At any given time, the regions indicated by red lines of β-end B is in contact with regions indicated by blue lines of β-end A. Similarly, the regions indicated by black lines of β-end B is in contact with regions indicated by green lines of β-end A. The first contact pair (red and blue) is seen for the entire duration of the simulation whereas the second contact pair (black and green) appears only towards end of the simulation. <b>D</b>) Time progression of contact regions between ACTH D and β-end B. At any given time, the regions indicated by red lines of ACTH D is in contact with regions indicated by blue lines of β-end B. Similarly, the regions indicated by black lines of β-end B is in contact with regions indicated by green lines of ACTH D. The first contact pair (red and blue) is seen for the initial part of the simulation whereas the second contact pair (green and black) appears only towards end of the simulation. Additional contacts are also observed for certain duration between the region of β-end B (aqua line) and ACTH D (pink line).</p

MD simulation of β-end system.

<p>All-atom MD simulation for the duration of 20 ns was performed in explicit solvent using four β-end. A) Snapshots indicating the initial (left) and final (right) states of the β-end system. B) Plot of distances between center of masses (Dij) of peptides within the β-end simulation against time. C) Contact map showing various residues in contact between peptides at t = 20 ns. Significant contacts are observed between amino acid residues in β-end A, β-end B and β-end D. D) A schematic depicting the various h-bonds observed between the peptides during the β-end simulation. The schematic showing C-terminus of β-end D is in contact with N-terminus of β-end A whereas C-terminus of β-end B is in contact with C-terminus β-end A. A salt bridge between E31 of β-end A and K28 of β-end B is represented by red dotted line.</p

Aggregation and colocalization of ACTH and β-end.

<p>A) Fluorescence photomicrographs of the anterior (A–C) and intermediate (D–F) lobes of the pituitary gland showing ACTH (A, D), β-end (B, E), and ACTH-β-end colocalized (C, F) cells. Note that presence of several double-labeled cells (arrows) in anterior as well as intermediate lobes. Scale bar = 50 µm. B) EM images of ACTH and β-end samples incubated for 14 days. The aggregations of the hormones were followed at 37°C at a concentration of 2 mg/ml in the presence of 0.4 mM LMW heparin in 5% D-mannitol (pH 5.5) and PBS under slight agitation. TEM of negative stained samples was performed. Scale bars, 500 nm.</p

Effect of N-terminal deletion on β-end aggregation.

<p>A) CD spectra of full-length β-end and β-end (6–31) at 2 mg/ml concentration in 5% D-mannitol, 0.01% sodium azide, pH 5.5 in presence and absence of 400 µM of LMW heparin. In the absence of heparin, both peptides remain mostly unstructured up to one week of incubation at 37°C. In presence of heparin, the full-length β-end showing structural transition from α-helix at d0 to mostly β-sheet at d4; while the β-end (6–31), which was in a mixed conformation of random coil and helix at d0, showing mostly α-helical conformation at d4. Both peptides show mostly β-sheet conformation at d7. (B) The ThT fluorescence study of one week incubated sample in presence of heparin showing less ThT binding of β-end (6–31) compared to full-length β-end. C) EM studies of 7 days incubated samples showing numerous amyloid-like fibrils for full-length β-end and fewer fibrils for β-end (6–31) in presence of heparin. Any amyloid-like fibrils are not observed in absence of heparin for both the peptides. D) AFM images of the full-length β-end and β-end (6–31) in presence and absence of heparin after a 7-day incubation showing more number of fibrils in case of the full length β-end sample as compared to the β-end (6–31) sample. Scale bars are of 500 nm.</p