Morphological Transformations of SARS-CoV-2 Nucleocapsid Protein Biocondensates Mediated by Antimicrobial Peptides.

Recently, the discovery of antimicrobial peptides (AMPs) as excellent candidates for overcoming antibiotic resistance has attracted significant attention. AMPs are short peptides active against bacteria, cancer cells, and viruses. It has been shown that the SARS-CoV-2 nucleocapsid protein (N-P) undergoes liquid-liquid phase separation in the presence of RNA, resulting in biocondensate formation. These biocondensates are crucial for viral replication as they concentrate the viral RNA with the host cell's protein machinery required for viral protein expression. Thus, N-P biocondensates are promising targets to block or slow down viral RNA transcription and consequently virion assembly. We investigated the ability of three AMPs to interfere with N-P/RNA condensates. Using microscopy techniques, supported by biophysical characterization, we found that the AMP LL-III partitions into the condensate, leading to clustering. Instead, the AMP CrACP1 partitions into the droplets without affecting their morphology but reducing their dynamics. Conversely, GKY20 leads to the formation of fibrillar structures after partitioning. It can be expected that such morphological transformation severely impairs the normal functionality of the N-P droplets and thus virion assembly. These results could pave the way for the development of a new class of AMP-based antiviral agents targeting biocondensates.

Clues to the Design of Aggregation-Resistant Insulin from Proline Scanning of Highly Amyloidogenic Peptides Derived from the N-Terminal Segment of the A-Chain.

Insulin aggregation poses a significant problem in pharmacology and medicine as it occurs during prolonged storage of the hormone and in vivo at insulin injection sites. We have recently shown that dominant forces driving the self-assembly of insulin fibrils are likely to arise from intermolecular interactions involving the N-terminal segment of the A-chain (ACC1-13). Here, we study how proline substitutions within the pilot GIVEQ sequence of this fragment affect its propensity to aggregate in both neutral and acidic environments. In a reasonable agreement with in silico prediction based on the Cordax algorithm, proline substitutions at positions 3, 4, and 5 turn out to be very effective in preventing aggregation according to thioflavin T-fluorescence-based kinetic assay, infrared spectroscopy, and atomic force microscopy (AFM). Since the valine and glutamate side chains within this segment are strongly involved in the interactions with the insulin receptor, we have focused on the possible implications of the Q → P substitution for insulin's stability and interactions with the receptor. To this end, comparative molecular dynamics (MD) simulations of the Q5P mutant and wild-type insulin were carried out for both free and receptor-bound (site 1) monomers. The results point to a mild destabilization of the mutant vis à vis the wild-type monomer, as well as partial preservation of key contacts in the complex between Q5P insulin and the receptor. We discuss the implications of these findings in the context of the design of aggregation-resistant insulin analogues retaining hormonal activity.

From a Droplet to a Fibril and from a Fibril to a Droplet: Intertwined Transition Pathways in Highly Dynamic Enzyme-Modulated Peptide-Adenosine Triphosphate Systems.

Many cellular coassemblies of proteins and polynucleotides facilitate liquid-liquid phase separation (LLPS) and the subsequent self-assembly of disease-associated amyloid fibrils within the liquid droplets. Here, we explore the dynamics of coupled phase and conformational transitions of model adenosine triphosphate (ATP)-binding peptides, ACC1-13Kn, consisting of the potent amyloidogenic fragment of insulin's A-chain (ACC1-13) merged with oligolysine segments of various lengths (Kn, n = 16, 24, 40). The self-assembly of ATP-stabilized amyloid fibrils is preceded by LLPS for peptides with sufficiently long oligolysine segments. The two-component droplets and fibrils are in dynamic equilibria with free ATP and monomeric peptides, which makes them susceptible to ATP-hydrolyzing apyrase and ACC1-13Kn-digesting proteinase K. Both enzymes are capable of rapid disassembly of amyloid fibrils, producing either monomers of the peptide (apyrase) or free ATP released together with cleaved-off oligolysine segments (proteinase K). In the latter case, the enzyme-sequestered Kn segments form subsequent droplets with the co-released ATP, resulting in an unusual fibril-to-droplet transition. In support of the highly dynamic nature of the aggregate-monomer equilibria, addition of superstoichiometric amounts of free peptide to the ACC1-13Kn-ATP coaggregate causes its disassembly. Our results show that the droplet state is not merely an intermediate phase on the pathway to the amyloid aggregate but may also constitute the final phase of a complex amyloidogenic protein misfolding scenario rich in highly degraded protein fragments incompetent to transition again into fibrils.

Inducin Triggers LC3-Lipidation and ESCRT-Mediated Lysosomal Membrane Repair.

Lipidation of the LC3 protein has frequently been employed as a marker of autophagy. However, LC3-lipidation is also triggered by stimuli not related to canonical autophagy. Therefore, characterization of the driving parameters for LC3 lipidation is crucial to understanding the biological roles of LC3. We identified a pseudo-natural product, termed Inducin, that increases LC3 lipidation independently of canonical autophagy, impairs lysosomal function and rapidly recruits Galectin 3 to lysosomes. Inducin treatment promotes Endosomal Sorting Complex Required for Transport (ESCRT)-dependent membrane repair and transcription factor EB (TFEB)-dependent lysosome biogenesis ultimately leading to cell death.

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, infrared spectroscopy, circular dichroism, and molecular dynamics simulations to study fibrillization patterns of two chimeric peptides, ACC1-13E8 and ACC1-13K8, in which a potent amyloidogenic stretch derived from the N-terminal segment of the insulin A-chain (ACC1-13) is coupled to octaglutamate or octalysine segments, respectively. While large electric charges prevent aggregation of either peptide 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 low-symmetry nonpolar ACC1-13 pilot sequence is crucial in enforcing the fibrillar structure consisting of parallel ß-sheets as the self-assembly of free poly-E and poly-K chains under similar conditions results in amorphous antiparallel ß-sheets. Interestingly, ACC1-13E8 forms highly ordered fibrils also when paired with nonpolypeptide polycationic amines such as branched polyethylenimine, instead of ACC1-13K8. Such synthetic polycations are more effective in triggering the fibrillization of ACC1-13E8 than poly-K (or poly-E in the case of ACC1-13K8). The 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 heterogeneous disease-related amyloidogenesis.

Liquid-Droplet-Mediated ATP-Triggered Amyloidogenic Pathway of Insulin-Derived Chimeric Peptides: Unraveling the Microscopic and Molecular Processes.

Disease-associated progression of protein dysfunction is typically determined by an interplay of transition pathways leading to liquid-liquid phase separation (LLPS) and amyloid fibrils. As LLPS introduces another layer of complexity into fibrillization of metastable proteins, a need for tunable model systems to study these intertwined processes has emerged. Here, we demonstrate the LLPS/fibrillization properties of a family of chimeric peptides, ACC1-13Kn, in which the highly amyloidogenic fragment of insulin (ACC1-13) is merged with oligolysine segments of various lengths (Kn, n = 8, 16, 24, 32, 40). LLPS and fibrillization of ACC1-13Kn are triggered by ATP through Coulombic interactions with Kn fragments. ACC1-13K8 and ACC1-13K16 form fibrils after a short lag phase without any evidence of LLPS. However, in the case of the three longest peptides, ATP triggers instantaneous LLPS followed by the disappearance of droplets occurring in-phase with the formation of amyloid fibrils. The kinetics of the phase transition and the stability of mature co-aggregates are highly sensitive to ionic strength, indicating that electrostatic interactions play a pivotal role in selecting the LLPS-fibrillization transition pathway. Densely packed ionic interactions that characterize ACC1-13Kn-ATP fibrils render them highly sensitive to hydrostatic pressure due to solvent electrostriction, as demonstrated by infrared spectroscopy. Using atomic force microscopy imaging of rapidly frozen samples, we demonstrate that early fibrils form within single liquid droplets, starting at the droplet/bulk interface through the formation of single bent fibers. A hypothetical molecular scenario underlying the emergence of the LLPS-to-fibrils pathway in the ACC1-13Kn-ATP system has been put forward.

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.

Aggregation of proteins into amyloid fibrils is driven by interactions between relatively small amyloidogenic segments. The interplay between aggregation-prone and aggregation-resistant fragments within a single polypeptide chain remains obscure. Here, we examine fibrillization behavior of two chimeric peptides, ACC1-13E8 and ACC1-13E8(L/D), in which the highly amyloidogenic fragment of insulin (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 ß-sheet while E8 forms antiparallel ß-sheets with the distinct infrared characteristics. This contrasts with the profoundly aggregation-resistant E8(L/D), although L/D patterns have been hypothesized as compatible with aggregated α-sheets. ACC1-13E8 and ACC1-13E8(L/D) are found to be equally prone to fibrillization at low pH, or in the presence of Ca2+ ions. Fibrillar states of both ACC1-13E8 and ACC1-13E8(L/D) reveal the infrared features of highly ordered parallel ß-sheet without evidence of ß2-aggregates (ACC1-13E8) or α-sheets (ACC1-13E8(L/D)). Hence, the preferred structural pattern of ACC1-13 overrides the tendency of E8 to form antiparallel ß-sheets and enforces the fibrillar order in E8(L/D). We demonstrate how the powerful amyloid stretch determines the overall amyloid structure forcing non-amyloidogenic fragments to participate in its native amyloid pattern.

Multiscale Modeling of Amyloid Fibrils Formed by Aggregating Peptides Derived from the Amyloidogenic Fragment of the A-Chain of Insulin.

Computational prediction of molecular structures of amyloid fibrils remains an exceedingly challenging task. In this work, we propose a multi-scale modeling procedure for the structure prediction of amyloid fibrils formed by the association of ACC1-13 aggregation-prone peptides derived from the N-terminal region of insulin's A-chain. First, a large number of protofilament models composed of five copies of interacting ACC1-13 peptides were predicted by application of CABS-dock coarse-grained (CG) docking simulations. Next, the models were reconstructed to all-atom (AA) representations and refined during molecular dynamics (MD) simulations in explicit solvent. The top-scored protofilament models, selected using symmetry criteria, were used for the assembly of long fibril structures. Finally, the amyloid fibril models resulting from the AA MD simulations were compared with atomic force microscopy (AFM) imaging experimental data. The obtained results indicate that the proposed multi-scale modeling procedure is capable of predicting protofilaments with high accuracy and may be applied for structure prediction and analysis of other amyloid fibrils.

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 a 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.

A tale of two tails: Self-assembling properties of A- and B-chain parts of insulin's highly amyloidogenic H-fragment.

Due to the spontaneous transition of native insulin into therapeutically inactive amyloid, prolonged storage decreases effectiveness of the hormone in treatment of diabetes. Various regions of the amino acid sequence have been implicated in insulin aggregation. Here, we focus on smaller fragments of the highly amyloidogenic H-peptide comprising disulfide-bonded N-terminal sections of insulin's A-chain (13 residues) and B-chain (11 residues). Aggregation patterns of N-terminal fragments of A-chain (ACC1-13, ACC1-11, ACC6-13, ACC6-11, all retaining Cys6A-Cys11A disulfide bond) and B-chain (B1-11(7A)) are examined at acidic and neutral pH. ACC1-11 is the smallest fragment found to be amyloidogenic at either pH; removal of the N-terminal GIVEQ section renders this fragment entirely non-amyloidogenic. The self-assembling properties of ACC1-11 contrast with aggregation-resistant behavior of B1-11(7A) and its disulfide-linked homodimer, (B1-11)2 aggregating only at neutral pH. Fibrillar ACC1-11 is similar to insulin amyloid in terms of morphology and infrared features. Secondary nucleation is likely to account for the detected shortening of insulin aggregation lag phase at neutral pH upon cross-seeding with pre-formed fibrils of ACC1-11 or (B1-11)2. An aggregation-enhancing effect of monomeric ACC1-11 on co-dissolved native insulin is also observed. Our findings are discussed in the context of mechanisms of insulin aggregation.

Neurotoxicity of oligomers of phosphorylated Tau protein carrying tauopathy-associated mutation is inhibited by prion protein.

Tauopathies, including Alzheimer's disease (AD), are manifested by the deposition of well-characterized amyloid aggregates of Tau protein in the brain. However, it is rather unlikely that these aggregates constitute the major form of Tau responsible for neurodegenerative changes. Currently, it is postulated that the intermediates termed as soluble oligomers, assembled on the amyloidogenic pathway, are the most neurotoxic form of Tau. However, Tau oligomers reported so far represent a population of poorly characterized, heterogeneous and unstable assemblies. In this study, to obtain the oligomers, we employed the aggregation-prone K18 fragment of Tau protein with deletion of Lys280 (K18Δ280) linked to a hereditary tauopathy. We have described a new procedure of inducing aggregation of mutated K18 which leads either to the formation of nontoxic amyloid fibrils or neurotoxic globular oligomers, depending on its phosphorylation status. We demonstrate that PKA-phosphorylated K18Δ280 oligomers are toxic to hippocampal neurons, which is manifested by loss of dendritic spines and neurites, and impairment of cell-membrane integrity leading to cell death. We also show that N1, the soluble N-terminal fragment of prion protein (PrP), protects neurons from the oligomers-induced cytotoxicity. Our findings support the hypothesis on the neurotoxicity of Tau oligomers and neuroprotective role of PrP-derived fragments in AD and other tauopathies. These observations could be useful in the development of therapeutic strategies for these diseases.

The Hunt for Ancient Prions: Archaeal Prion-Like Domains Form Amyloid-Based Epigenetic Elements.

Prions, proteins that can convert between structurally and functionally distinct states and serve as non-Mendelian mechanisms of inheritance, were initially discovered and only known in eukaryotes, and consequently considered to likely be a relatively late evolutionary acquisition. However, the recent discovery of prions in bacteria and viruses has intimated a potentially more ancient evolutionary origin. Here, we provide evidence that prion-forming domains exist in the domain archaea, the last domain of life left unexplored with regard to prions. We searched for archaeal candidate prion-forming protein sequences computationally, described their taxonomic distribution and phylogeny, and analyzed their associated functional annotations. Using biophysical in vitro assays, cell-based and microscopic approaches, and dye-binding analyses, we tested select candidate prion-forming domains for prionogenic characteristics. Out of the 16 tested, eight formed amyloids, and six acted as protein-based elements of information transfer driving non-Mendelian patterns of inheritance. We also identified short peptides from our archaeal prion candidates that can form amyloid fibrils independently. Lastly, candidates that tested positively in our assays had significantly higher tyrosine and phenylalanine content than candidates that tested negatively, an observation that may help future archaeal prion predictions. Taken together, our discovery of functional prion-forming domains in archaea provides evidence that multiple archaeal proteins are capable of acting as prions-thus expanding our knowledge of this epigenetic phenomenon to the third and final domain of life and bolstering the possibility that they were present at the time of the last universal common ancestor.

Extremely Amyloidogenic Single-Chain Analogues of Insulin's H-Fragment: Structural Adaptability of an Amyloid Stretch.

Relatively short amino acid sequences often play a pivotal role in triggering protein aggregation leading to the formation of amyloid fibrils. In the case of insulin, various regions of A- and B-chains have been implicated as the most relevant to the protein's amyloidogenicity. Here, we focus on the highly amyloidogenic H-fragment of insulin comprising the disulfide-bonded N-terminal parts of both chains. Analysis of the aggregation behavior of single-chain peptide derivatives of the H-fragment suggests that the A-chain's part initiates the aggregation process while the disulfide-tethered B-chain reluctantly adapts to amyloid structure. Merging of both A- and B-parts into single-chain continuous peptides (A-B and B-A) results in extreme amyloidogenicity exceeding that of the double-chain H-fragment as reflected by almost instantaneous de novo fibrillization. Amyloid fibrils of A-B and B-A present distinct morphological and infrared traits and do not cross-seed insulin. Our study suggests that the N-terminal part of insulin's A-chain containing the intact Cys6-Cys11 intrachain disulfide bond may constitute insulin's major amyloid stretch which, through its bent conformation, enforces a parallel in-register alignment of ß-strands. Comparison of the self-association behavior of H, A-B, and B-A peptides suggests that A-chain's N-terminal amyloid stretch is very versatile and adaptive to various structural contexts.

Reduction of a disulfide-constrained oligo-glutamate peptide triggers self-assembly of ß2-type amyloid fibrils with the chiroptical properties determined by supramolecular chirality.

Disulfide bonds prevent aggregation of globular proteins by stabilizing the native state. However, a disulfide bond within a disordered state may accelerate amyloidogenic nucleation by navigating fluctuating polypeptide chains towards an orderly assembly of ß-sheets. Here, the self-assembly behavior of Glu-Cys-(Glu)4-Cys-Glu peptide (E6C2), in which an intrachain disulfide bond is engineered into an amyloidogenic homopolypeptide motif, is investigated. To this end, the Thioflavin T (ThT) fluorescence kinetic assay is combined with infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and Raman scattering measurements. Regardless of whether the disulfide bond is intact or reduced, E6C2 monomers remain disordered within a broad range of pH. On the other hand, only reduced E6C2 self-assembles into amyloid fibrils with the unique infrared traits indicative of three-center hydrogen bonds involving main-chain carbonyl as a bifurcating acceptor and main-chain NH and side-chain -COOH groups as hydrogen donors: the bonding pattern observed in so-called ß2-fibrils. AFM analysis of ß2-E6C2 reveals tightly packed rectangular superstructures whose presence coincides with strong chiroptical properties. Our findings suggest that formation of chiral amyloid superstructures may be a generic process accessible to various substrates, and that the fully extended conformation of a poly-Glu chain is a condition sine qua non for self-assembly of ß2-fibrils.

Docking interactions determine early cleavage events in insulin proteolysis by pepsin: Experiment and simulation.

In silico modelling of cascade enzymatic proteolysis is an exceedingly complex and challenging task. Here, we study partial proteolysis of insulin by pepsin: a process leading to the release of a highly amyloidogenic two chain 'H-fragment'. The H-fragment retains several cleavage sites for pepsin. However, under favorable conditions H-monomers rapidly self-assemble into proteolysis-resistant amyloid fibrils whose composition provides snapshots of early and intermediate stages of the proteolysis. In this work, we report a remarkable agreement of experimentally determined and simulation-predicted cleavage sites on different stages of the proteolysis. Prediction of cleavage sites was based on the comprehensive analysis of the docking interactions from direct simulation of coupled folding and binding of insulin (or its cleaved derivatives) to pepsin. The most frequent interactions were found to be between the pepsin's active site, or its direct vicinity, and the experimentally determined insulin cleavage sites, which suggest that the docking interactions govern the proteolytic process.

Rapid self-association of highly amyloidogenic H-fragments of insulin: Experiment and molecular dynamics simulations.

The so-called 'H-fragment' of insulin is an extremely amyloidogenic double chain peptide consisting of the N-terminal parts of A-chain and B-chain linked by a disulfide bond between Cys-7A and Cys-7B. Here, we conduct a detailed investigation of the self-association behavior of H-fragment monomers into amyloid-like fibrils using kinetic assays, infrared spectroscopy, circular dichroism (CD), atomic force microscopy (AFM) and molecular dynamics (MD) simulations. Unlike the intact predominantly α-helical insulin, H-fragment remains in a disordered state in aqueous solutions. Its aggregation accelerates with acidification of the environment leading, at pH 1.9, to the formation of thin and structurally homogenous fibrils with the infrared features typical for parallel ß-sheet conformation. According to time-lapse AFM morphological analysis both secondary nucleation and fragmentation are involved in later stages of H-fibrils' self-assembly. Based on the low nucleation order (two) obtained from the global fitting of kinetic data, realistic all-atom MD simulations of pairs of interacting H-fragment monomers were subsequently carried out. The molecular self-association scenario emerging from these simulations implicates the intrinsic conformational instability of H-monomer in its tendency to aggregate and form intermolecular ß-sheet structure. Our findings provide the new mechanistic context for studies of insulin misfolding and aggregation.

Reversible Freeze-Induced ß-Sheet-to-Disorder Transition in Aggregated Homopolypeptide System.

Conformational transitions involving aggregated proteins or peptides are of paramount biomedical and biotechnological importance. Here, we report an unusual freeze-induced structural reorganization within a ß-sheet-rich ionic coaggregate of poly(l-lysine), PLL, and poly(l-glutamic acid), PLGA. Freezing aqueous suspensions of the PLL-PLGA ß-aggregate in the presence of low concentrations of salt (NaBr) induces an instantaneous ß-sheet-to-disorder transition, as probed by infrared spectroscopy in the amide I' band region. The conformational rearrangement of polypeptide chains appears to be fully synchronized with the global liquid-to-ice phase transition. In contrast to the known freeze-induced transitions, the process described here is fully reversible: the subsequent thawing results in an instantaneous disorder-to-ß-sheet "refolding". However, in the absence of traces of soluble salts, the ß-sheet framework of the PLL-PLGA aggregate remains resistant to freezing as no transition is observed. We note that the occurrence of the transition depends on the type of salt present in the sample. Our results highlight a hidden dimension of the structural dynamics within ß-sheet-rich aggregates. Possible scenarios of freeze-induced salt-bridge rupture and removal of water from nanocanals are discussed.

Revisiting the conformational state of albumin conjugated to gold nanoclusters: A self-assembly pathway to giant superstructures unraveled.

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native ß-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.

Beyond amino acid sequence: disulfide bonds and the origins of the extreme amyloidogenic properties of insulin's H-fragment.

The presence of disulfide bonds affects the protein stability and therefore tendency to misfold and form amyloid-like fibrils. Insulin's three disulfide bridges stabilize the native state and prevent aggregation. Partial proteolysis of insulin releases highly amyloidogenic and inherently disordered two-chain 'H-fragment' retaining insulin's Cys7A-Cys7B and Cys6A-Cys11A disulfide bonds. The abrupt self-association of H-fragment monomers into fibrils is suppressed in the presence of disulfide-reducing agent. These circumstances make the H-fragment an interesting model to study the impact of disulfide bonds on amyloidogenesis beyond the 'stabilization-of-the-native-state' paradigm. Here, we investigate fibrillization of various synthetic peptides derived from the H-fragment through modifications of Cys7A-Cys7B/Cys6A-Cys11A bonds. In comparison to H-fragment, aggregation of a two-chain 'AB' analog lacking Cys6A-Cys11A bond is decelerated, while the alternative removal of Cys7A-Cys7B bond releases a non-aggregating B-chain and a highly amyloidogenic 'ACC' fragment containing the intrachain Cys6A-Cys11A bond. Our analysis, supported by calculations of configurational entropy, suggests that Cys6A-Cys11A bond is a key factor behind the explosive self-association of H-fragment. The bond restricts the conformational space probed by nucleating monomers which is reflected by an approximately 2.4 kJ·mol-1 K-1 decrease in entropy. The fact that the intact Cys6A-Cys11A bond promotes fibrillization of the H-fragment is remarkable in light of the previously established role of the same disulfide bond in preventing formation of insulin fibrils. Our results imply that a single disulfide bond within a folded protein and its fragment may play entirely different roles in aggregation and that this role may evolve with progressing phases of misfolding.

ß2-Type Amyloidlike Fibrils of Poly-l-glutamic Acid Convert into Long, Highly Ordered Helices upon Dissolution in Dimethyl Sulfoxide.

Replacing water with dimethyl sulfoxide (DMSO) completely reshapes the free-energy landscapes of solvated proteins. In DMSO, a powerful hydrogen-bond (HB) acceptor, formation of HBs between backbone NH groups and solvent is favored over HBs involving protein's carbonyl groups. This entails a profound structural disruption of globular proteins and proteinaceous aggregates (e.g., amyloid fibrils) upon transfer to DMSO. Here, we investigate an unusual DMSO-induced conformational transition of ß2-amyloid fibrils from poly-l-glutamic acid (PLGA). The infrared spectra of ß2-PLGA dissolved in DMSO lack the typical features associated with disordered conformation that are observed when amyloid fibrils from other proteins are dispersed in DMSO. Instead, the frequency and unusual narrowness of the amide I band imply the presence of highly ordered helical structures, which is supported by complementary methods, including vibrational circular dichroism and Raman optical activity. We argue that the conformation most consistent with the spectroscopic data is that of a PLGA chain essentially lacking nonhelical segments such as bends that would provide DMSO acceptors with direct access to the backbone. A structural study of DMSO-dissolved ß2-PLGA by synchrotron small-angle X-ray scattering reveals the presence of long uninterrupted helices lending direct support to this hypothesis. Our study highlights the dramatic effects that solvation may have on conformational transitions of large polypeptide assemblies.