Virtual quasi-2D intermediates as building blocks for plausible structural models of amyloid fibrils from proteins with complex topologies: A case study of insulin

Conformational transitions of globular proteins into amyloid fibrils are complex multistage processes exceedingly challenging to simulate using molecular dynamics (MD). Slow monomer diffusion rates and rugged free energy landscapes disfavor swift self-assembly of orderly amyloid architectures within timescales accessible to all-atom MD. Here, we conduct a multiscale MD study of the amyloidogenic self-assembly of insulin: a small protein with complex topology defined by two polypeptide chains interlinked by three disulfide bonds. In order to avoid kinetic traps, unconventional pre-planarized insulin conformations are used as amyloid building blocks. These starting conformers generated through uniaxial compression of the native monomer in various spatial directions represent 6 distinct (out of 16 conceivable) 2D topological classes varying in N- / C-terminal segments of insulin’s A- and B-chains being placed inside, or outside of the central loop constituted by the middle sections of both chains and Cys7A-Cys7B / Cys19B-Cys20A disulfide bonds. Simulations of the fibrillar self-assembly are initiated through a biased in-register alignment of 2, 3 or 4 layers of flat conformers belonging to a single topological class. The various starting topologies are conserved throughout the self-assembly process resulting in polymorphic amyloid fibrils varying in structural features such as helical twist, presence of cavities, as well as the overall stability. Some of the protofilament structures obtained in this work are highly compatible with the earlier biophysical studies on insulin amyloid and high resolution studies on insulin-derived amyloidogenic peptide models postulating presence of steric zippers. Our approach provides in silico means to study amyloidogenic tendencies and viable amyloid architectures of larger disulfide-constrained proteins with complex topologies

Enzymatic digestion of luminescent albumin-stabilized gold nanoclusters under anaerobic conditions: clues to the quenching mechanism

Many of the potential applications of albumin-stabilized gold nanoclusters (AuNC) arise from the sensitivity of their luminescence to the presence of various ions and albumin-degrading proteases. However, the underlying photophysics and the mechanisms responsible for protease-induced quenching of AuNC luminescence are not fully understood. Here, we study proteinase K-induced digestion of bovine serum albumin (BSA)-AuNC conjugate under aerobic and anaerobic conditions. To this end, we adapt a Co(II)-catalyzed sulfite-based protocol enabling effective in situ deoxidization without deactivation of the enzyme. In the absence of proteinase K, the anaerobic conditions facilitate luminescence of BSA-AuNC reflected by a moderate increase in the red luminescence intensity. However, in the presence of proteinase K, we have observed a steeper decrease of emission intensity irrespective of whether the digestion was carried out under aerobic or anaerobic conditions. In both cases, the diminishing fluorescence occurred in phase with shifting of the emission maximum to longer wavelengths. These results contradict the previous hypothesis that protease-induced quenching of BSA-AuNC luminescence is a consequence of enhanced diffusion of oxygen to bare AuNC. Instead, aggregation of unprotected AuNCs and separation of nanoclusters from albumin’s side chains involved in energy transfers and luminescence-promoting electron donors may underlie the observed sensitivity of BSA-AuNC to protease treatment. Our findings are discussed in the context of mechanisms of formation and photophysics of BSA-AuNC conjugates

Selective and stoichiometric incorporation of ATP by self-assembling amyloid fibrils.

ATP acts as a biological hydrotrope preventing protein aggregation. Here, we report a novel chimeric peptide, ACC1-13K8, with an unusual capacity to bind and incorporate ATP while self-assembling into amyloid fibrils. The amino acid sequence combines highly amyloidogenic segment of insulin’s A-chain (ACC1-13) and octalysine (K8). Fibrillization requires binding 2 ATP molecules per ACC1-13K8 monomer and is not triggered by adenosine di- and monophosphates (ADP, AMP). Infrared and CD spectra and AFM-based morphological analysis reveal tight and orderly entrapment of ATP within superstructural hybrid peptide-ATP fibrils. The incorporation of ATP is an emergent property of ACC1-13K8 not observed for ACC1-13 and K8 segments separately. We demonstrate how new functionalities (e.g. ATP storage) emerge from synergistic coupling of amyloidogenic segments with non-amyloidogenic peptide ligands, and suggest that ATP’s role in protein misfolding is more nuanced than previously assumed

Forced amyloidogenic cooperativity of structurally incompatible peptide segments: Fibrillization behavior of highly aggregation-prone A-chain fragment of insulin coupled to all-L, and alternating L/D octaglutamates

Self-aggregation of individual polypeptide chains into amyloid fibrils is driven by interactions between amyloidogenic segments of whole proteins. The interplay between aggregation-prone and aggregation-resistant fragments within a single polypeptide chain is not well understood. Here, we examine fibrillization behavior of two designed chimeric peptides, ACC1-13E8 and ACC1-13E8(L/D), in which the highly amyloidogenic fragment of insulin’s A-chain (ACC1-13) is extended by an octaglutamate segment composed of all-L (E8), or alternating L/D residues (E8(L/D)). As separate entities, ACC1-13 readily forms fibrils with the infrared features of parallel β-sheet structure while acidified E8 forms so-called β2-aggregates consisting of antiparallel β-sheets and manifesting distinctly in the amide I band infrared region. This contrasts with profoundly aggregation-resistant behavior of E8(L/D) peptide although the alternating L/D motif has been hypothesized as compatible with aggregated α-sheets. ACC1-13E8 and ACC1-13E8(L/D) peptides are equally prone to fibrillization when the electrostatic repulsion between dissolved monomers is prevented either by lowering pH, or in the presence of Ca2+ ions. In the aggregated states, both ACC1-13E8 and ACC1-13E8(L/D) reveal the infrared characteristics of ordered parallel β-sheet structure with no spectral features attributable to β2-aggregates (ACC1-13E8) or α-sheets (ACC1-13E8(L/D)). Hence, the preferred structural pattern of ACC1-13 segment not only overrides the tendency of E8 to form the antiparallel β2-structure but also enforces formation of β-sheet structure within the E8(L/D) segment which on its own is entirely refractory to aggregation. We demonstrate how an alternating L/D sequence can be effectively forced to become a part of highly ordered amyloid structure scaffolded by an all-L amyloidogenic segment. Our study shows how the overall amyloidogenic characteristics of a larger hybrid sequence may be impacted and controlled by the properties of its most aggregation-prone part

Self-assembly of insulin-derived chimeric peptides into two-component amyloid fibrils: the role of Coulombic interactions

Canonical amyloid fibrils are composed of covalently identical polypeptide chains. Here, we employ kinetic assays, atomic force microscopy (AFM), infrared spectroscopy, circular dichroism (CD), and molecular dynamics (MD) to study fibrillization patterns of two chimeric peptides, ACC1-13E8 and ACC1-13K8, in which potent amyloidogenic stretch derived from the N-terminal segment of insulin A-chain (ACC1-13) is coupled to octaglutamate or octalysine segments, respectively. While the large electric charges on monomers of either peptide prevent aggregation at neutral pH, stoichiometric mixing of ACC1-13E8 and ACC1-13K8 triggers rapid self-assembly of two-component fibrils driven by favorable Coulombic interactions. The role of low-symmetry non-polar ACC1-13 pilot sequence is crucial in enforcing the amyloidal parallel -sheet motif as self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel -sheet conformation. Interestingly, the pathway to highly ordered fibrils is accessible to ACC1-13E8 also when paired with non-polypeptide polycationic amines such as branched poly-ethylenimine, PEI, instead of ACC1-13K8. Remarkably, such synthetic polycations are more effective in triggering fibrillization of ACC1-13E8 than poly-K (or poly-E in the case of ACC1-13K8). High conformational flexibility of these polyamines makes up for the apparent mismatch in periodicity of charged groups. The results are discussed in the context of mechanisms of heterogenous disease-related amyloidogenesis

Hidden dynamics of noble-metal-bound thiol monolayers revealed by SERS-monitored entropy-driven exchange of cysteine isotopologues

Vibrational spectroscopy coupled with isotopic labeling provides many insights into dynamic processes within various molecular systems. Here, a newfound utility of surface-enhanced Raman scattering (SERS) spectroscopy as a tool to study noble metal-anchored thiol monolayers is demonstrated for a pair of L-cysteine isotopologues competing to bind the surface of silver nanoparticles (AgNPs). According to our DFT calculations, SERS spectra of L-Cys could be sensitive to 12C/13C and 14N/15N isotopic substitutions, which has been experimentally confirmed for the pair of L-Cys isotopologues: Cys-cabn (all 12C/14N) and Cys-CABN (all 13C/15N). In the AgNP-anchored state, the two isotopologues reveal distinct Raman shift values (1577 cm-1 / 1633 cm-1) of the band assigned to C=O stretching. This characteristic SERS feature has been subsequently employed to probe various exchange scenarios between AgNP-bound and free L-Cys molecules. As the exchange involves two spectrally distinct but chemically identical molecules, the process is exclusively entropy-driven ultimately leading to the equilibrium state in which Cys-cabn/Cys-CABN concentration ratios in the Ag surface-bound layers and the bulk solution are identical. In a system containing AgNP-L-Cys and an excess of free L-Cys molecules, the exchange energy barrier limits the overall kinetics. Although the SERS-monitored rate of progression toward the equilibrium state under ambient conditions (25 °C) is negligible, a very steep acceleration of the exchange is observed at 50 °C. While the temperature-induced transition is very abrupt, it is still reversible with cooling. We argue that the dramatic acceleration of the dynamics of the L-Cys exchange between free and AgNP-bound molecules may be rationalized as a collective phase transition to an excited and reaction-prone state. Our work highlights unexplored potential of SERS spectroscopy coupled to isotopic exchange as a tool to study obscured dynamic phenomena within metal-anchored adsorbate layers

Covalent defects restrict supramolecular self-assembly of homopolypeptides: case study of β2-fibrils of poly-L-glutamic acid.

Poly-L-glutamic acid (PLGA) often serves as a model in studies on amyloid fibrils and conformational transitions in proteins, and as a precursor for synthetic biomaterials. Aggregation of PLGA chains and formation of amyloid-like fibrils was shown to continue on higher levels of superstructural self-assembly coinciding with the appearance of so-called β2-sheet conformation manifesting in dramatic redshift of infrared amide I' band below 1600 cm(-1). This spectral hallmark has been attributed to network of bifurcated hydrogen bonds coupling C = O and N-D (N-H) groups of the main chains to glutamate side chains. However, other authors reported that, under essentially identical conditions, PLGA forms the conventional in terms of infrared characteristics β1-sheet structure (exciton-split amide I' band with peaks at ca. 1616 and 1683 cm(-1)). Here we attempt to shed light on this discrepancy by studying the effect of increasing concentration of intentionally induced defects in PLGA on the tendency to form β1/β2-type aggregates using infrared spectroscopy. We have employed carbodiimide-mediated covalent modification of Glu side chains with n-butylamine (NBA), as well as electrostatics-driven inclusion of polylysine chains, as two different ways to trigger structural defects in PLGA. Our study depicts a clear correlation between concentration of defects in PLGA and increasing tendency to depart from the β2-structure toward the one less demanding in terms of chemical uniformity of side chains: β1-structure. The varying predisposition to form β1- or β2-type aggregates assessed by infrared absorption was compared with the degree of morphological order observed in electron microscopy images. Our results are discussed in the context of latent covalent defects in homopolypeptides (especially with side chains capable of hydrogen-bonding) that could obscure their actual propensities to adopt different conformations, and limit applications in the field of synthetic biomaterials