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Background: Frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-TDP), amyotrophic lateral sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE) are associated with deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43) in neurons. One complexity of this process lies in the ability of TDP-43 to form liquid-phase membraneless organelles in cells. Previous work has shown that the recombinant, purified, prion-like domain (PrLD) forms liquid droplets in vitro, but the behaviour of the complementary fragment is uncertain., Methods: We have purified such a construct without the PrLD (PrLD-less TDP-43) and have induced its phase separation using a solution-jump method and an array of biophysical techniques to study the morphology, state of matter and structure of the TDP-43 assemblies., Results: The fluorescent TMR-labelled protein construct, imaged using confocal fluorescence, formed rapidly (< 1 min) round, homogeneous and 0.5-1.0 µm wide assemblies which then coalesced into larger, yet round, species. When labelled with AlexaFluor488, they initially exhibited fluorescence recovery after photobleaching (FRAP), showing a liquid behaviour distinct from full-length TDP-43 and similar to PrLD. The protein molecules did not undergo major structural changes, as determined with circular dichroism and intrinsic fluorescence spectroscopies. This process had a pH and salt dependence distinct from those of full-length TDP-43 and its PrLD, which can be rationalized on the grounds of electrostatic forces., Conclusions: Similarly to PrLD, PrLD-less TDP-43 forms liquid droplets in vitro through liquid-liquid phase separation (LLPS), unlike the full-length protein that rather undergoes liquid-solid phase separation (LSPS). These results offer a rationale of the complex electrostatic forces governing phase separation of full-length TDP-43 and its fragments. On the one hand, PrLD-less TDP-43 has a low pI and oppositively charged domains, and LLPS is inhibited by salts, which attenuate inter-domain electrostatic attractions. On the other hand, PrLD is positively charged due to a high isoionic point (pI) and LLPS is therefore promoted by salts and pH increases as they both reduce electrostatic repulsions. By contrast, full-length TDP-43 undergoes LSPS most favourably at its pI, with positive and negative salt dependences at lower and higher pH, respectively, depending on whether repulsive or attractive forces dominate, respectively., (© 2024. The Author(s).)

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Natural aminosterols are promising drug candidates against neurodegenerative diseases, like Alzheimer and Parkinson, and one relevant protective mechanism occurs via their binding to biological membranes and displacement or binding inhibition of amyloidogenic proteins and their cytotoxic oligomers. We compared three chemically different aminosterols, finding that they exhibited different (i) binding affinities, (ii) charge neutralizations, (iii) mechanical reinforcements, and (iv) key lipid redistributions within membranes of reconstituted liposomes. They also had different potencies (EC 50 ) in protecting cultured cell membranes against amyloid-β oligomers. A global fitting analysis led to an analytical equation describing quantitatively the protective effects of aminosterols as a function of their concentration and relevant membrane effects. The analysis correlates aminosterol-mediated protection with well-defined chemical moieties, including the polyamine group inducing a partial membrane-neutralizing effect (79 ± 7%) and the cholestane-like tail causing lipid redistribution and bilayer mechanical resistance (21 ± 7%), linking quantitatively their chemistry to their protective effects on biological membranes.

TAR DNA-binding protein 43 (TDP-43) forms intraneuronal cytoplasmic inclusions associated with amyotrophic lateral sclerosis and ubiquitin-positive frontotemporal lobar degeneration. Its N-terminal domain (NTD) can dimerise/oligomerise with the head-to-tail arrangement, which is essential for function but also favours liquid-liquid phase separation and inclusion formation of full-length TDP-43. Using various biophysical approaches, we identified an alternative conformational state of NTD in the presence of Sulfobetaine 3-10 (SB3-10), with higher content of α-helical structure and tryptophan solvent exposure. NMR shows a highly mobile structure, with partially folded regions and β-sheet content decrease, with a concomitant increase of α-helical structure. It is monomeric and reverts to native oligomeric NTD upon SB3-10 dilution. The equilibrium GdnHCl-induced denaturation shows a cooperative folding and a somewhat lower conformational stability. When the aggregation processes were compared with and without pre-incubation with SB3-10, but at the identical final SB3-10 concentration, a slower aggregation was found in the former case, despite the reversible attainment of the native conformation in both cases. This was attributed to protein monomerization and oligomeric seeds disruption by the conditions promoting the alternative conformation. Overall, the results show a high plasticity of TDP-43 NTD and identify strategies to monomerise TDP-43 NTD for methodological and biomedical applications.

The release of monomers from the homotetrameric protein transthyretin (TTR) is the first event of a cascade, eventually leading to sporadic or familial TTR amyloidoses. Thus, ligands able to stabilize TTR and inhibit monomer release are subject of intense scrutiny as potential treatments against these pathologies. Here, we investigated the interaction between TTR and a non-glycated derivative of the main olive polyphenol, oleuropein (OleA), known to interfere with TTR aggregation. We coupled fluorescence studies with molecular docking to investigate the OleA/TTR interaction using wild-type TTR, a monomeric variant, and the L55P cardiotoxic mutant. We characterized a fluorescence band emitted by OleA upon formation of the OleA/TTR complex. Exploiting this signal, we found that a poorly specific non-stoichiometric interaction occurs on the surface of the protein and a more specific stabilizing interaction takes place in the ligand binding pocket of TTR, exhibiting a K D of 3.23 ± 0.32 µM, with two distinct binding sites. OleA interacts with TTR in different modes, stabilizing it and preventing its dissociation into monomers, with subsequent misfolding. This result paves the way to the possible use of OleA to prevent degenerative diseases associated with TTR misfolding.

TAR DNA-binding protein 43 (TDP-43) is a 414-residue long nuclear protein whose deposition into intraneuronal insoluble inclusions has been associated with the onset of amyotrophic lateral sclerosis (ALS) and other diseases. This protein is physiologically a homodimer, and dimerization occurs through the N-terminal domain (NTD), with a mechanism on which a full consensus has not yet been reached. Furthermore, it has been proposed that this domain is able to affect the formation of higher molecular weight assemblies. Here, we purified this domain and carried out an unprecedented characterization of its folding/dimerization processes in solution. Exploiting a battery of biophysical approaches, ranging from FRET to folding kinetics, we identified a head-to-tail arrangement of the monomers within the dimer. We found that folding of NTD proceeds through the formation of a number of conformational states and two parallel pathways, while a subset of molecules refold slower, due to proline isomerism. The folded state appears to be inherently prone to form high molecular weight assemblies. Taken together, our results indicate that NTD is inherently plastic and prone to populate different conformations and dimeric/multimeric states, a structural feature that may enable this domain to control the assembly state of TDP-43., Competing Interests: The authors declare no conflict of interest.

Alzheimer's disease is associated with the deposition of the amyloid-β peptide (Aβ) into extracellular senile plaques in the brain. In vitro and in vivo observations have indicated that transthyretin (TTR) acts as an Aβ scavenger in the brain, but the mechanism has not been fully resolved. We have monitored the aggregation process of Aβ 40 by thioflavin T fluorescence, in the presence or absence of different concentrations of preformed seed aggregates of Aβ 40 , of wild-type tetrameric TTR (WT-TTR), and of a variant engineered to be stable as a monomer (M-TTR). Both WT-TTR and M-TTR were found to inhibit specific steps of the process of Aβ 40 fibril formation, which are primary and secondary nucleations, without affecting the elongation of the resulting fibrils. Moreover, the analysis shows that both WT-TTR and M-TTR bind to Aβ 40 oligomers formed in the aggregation reaction and inhibit their conversion into the shortest fibrils able to elongate. Using biophysical methods, TTR was found to change some aspects of its overall structure following such interactions with Aβ 40 oligomers, as well as with oligomers of Aβ 42 , while maintaining its overall topology. Hence, it is likely that the predominant mechanism by which TTR exerts its protective role lies in the binding of TTR to the Aβ oligomers and in inhibiting primary and secondary nucleation processes, which limits both the toxicity of Aβ oligomers and the ability of the fibrils to proliferate.

Deposition of soluble proteins as insoluble amyloid fibrils is associated with a number of pathological states. There is a growing interest in the identification of small molecules that can prevent proteins from undergoing amyloid fibril formation. In the present study, a series of small aromatic compounds with different substitutions of 1,3,5-triphenylbenzene have been synthesized and their possible effects on amyloid fibril formation by hen egg white lysozyme (HEWL), a model protein for amyloid formation, and of their resulting toxicity were examined. The inhibitory effect of the compounds against HEWL amyloid formation was analyzed using thioflavin T and Congo red binding assays, atomic force microscopy, Fourier-transform infrared spectroscopy, and cytotoxicity assays, such as the 3-(4,5-Dimethylthiazol)-2,5-Diphenyltetrazolium Bromide (MTT) reduction assay and caspase-3 activity measurements. We found that all compounds in our screen were efficient inhibitors of HEWL fibril formation and their associated toxicity. We showed that electron-withdrawing substituents such as -F and -NO 2 potentiated the inhibitory potential of 1,3,5-triphenylbenzene, whereas electron-donating groups such as -OH, -OCH 3 , and -CH 3 lowered it. These results may ultimately find applications in the development of potential inhibitors against amyloid fibril formation and its biologically adverse effects.

We have studied the intrinsic fluorescence spectra of a monomeric variant of human transthyretin (M-TTR), a protein involved in the transport of the thyroid hormone and retinol and associated with various forms of amyloidosis, extending our analysis to the second order derivative of the spectra. This procedure allowed to identify three peaks readily assigned to Trp41, as the three peaks were also visible in a mutant lacking the other tryptophan (Trp79) and had similar FRET efficiency values with an acceptor molecule positioned at position 10. The wavelength values of the three peaks and their susceptibility to acrylamide quenching revealed that the three corresponding conformers experience different solvent-exposure, polarity of the environment and flexibility. We could monitor the three peaks individually in urea-unfolding and pH-unfolding curves. This revealed changes in the distribution of the corresponding conformers, indicating conformational changes and alterations of the dynamics of the microenvironment that surrounds the associated tryptophan residue in such transitions, but also native-like conformers of such residues in unfolded states. We also found that the amyloidogenic state adopted by M-TTR at mildly low pH has a structural and dynamical microenvironment surrounding Trp41 indistinguishable from that of the fully folded and soluble state at neutral pH.

Novel imaging techniques with ever-increasing resolution are invaluable tools for the study of protein deposition, as they allow the self-assembly of proteins to be directly investigated in living cells. For the first time, the acceleration in Aβ42 aggregation induced by the Arctic mutation was monitored in cells, revealing a number of distinct morphologies that form sequentially. This approach will help discriminate the impacts of mutations on amyloid protein processing, Aβ aggregation propensity, and other mechanistic outcomes., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2019 Bemporad et al.)

A set of missense mutations in the gene encoding profilin-1 has been linked to the onset of familial forms of ALS (fALS), also known as Lou Gehrig's disease. The pathogenic potential of these mutations is linked to the formation of intracellular inclusions of the mutant proteins and correlates with the mutation-induced destabilization of its native, fully folded state. However, the mechanism by which these mutations promote misfolding and self-assembly is yet unclear. Here, using temperature-jump and stopped-flow kinetic measurements, we show that, during refolding, WT profilin-1 transiently populates a partially folded (PF) state endowed with hydrophobic clusters exposed to the solvent and with no detectable secondary structure. We observed that this conformational state is marginally stable at neutral pH but becomes significantly populated at mildly acidic pH. Interestingly, the fALS-associated mutations did not cause a change in the refolding mechanism of profilin-1, but induced a stabilization of the PF state. In the presence of preformed profilin-1 aggregates, the PF state, unlike the unfolded and folded states, could interact with these aggregates via nonspecific hydrophobic interactions and also increase thioflavin-T fluorescence, revealing its amyloidogenic potential. Moreover, in the variants tested, we found a correlation between conformational stability of PF and aggregation propensity, defining this conformational state as an aggregation-prone folding intermediate. In conclusion, our findings indicate that mutation-induced stabilization of a partially folded state can enhance profilin-1 aggregation and thereby contribute to the pathogenicity of the mutations., (© 2018 Del Poggetto et al.)

Transthyretin (TTR) is an extracellular protein able to deposit into well-defined protein aggregates called amyloid, in pathological conditions known as senile systemic amyloidosis, familial amyloid polyneuropathy, familial amyloid cardiomyopathy and leptomeningeal amyloidosis. At least three distinct partially folded states have been described for TTR, including the widely studied amyloidogenic state at mildly acidic pH. Here, we have used fluorescence resonance energy transfer (FRET) experiments in a monomeric variant of TTR (M-TTR) and in its W41F and W79F mutants, taking advantage of the presence of a unique, solvent-exposed, cysteine residue at position 10, that we have labelled with a coumarin derivative (DACM, acceptor), and of the two natural tryptophan residues at positions 41 and 79 (donors). Trp41 is located in an ideal position as it is one of the residues of β-strand C, whose degree of unfolding is debated. We found that the amyloidogenic state at low pH has the same FRET efficiency as the folded state at neutral pH in both M-TTR and W79F-M-TTR, indicating an unmodified Cys10-Trp41 distance. The partially folded state populated at low denaturant concentrations also has a similar FRET efficiency, but other spectroscopic probes indicate that it is distinct from the amyloidogenic state at acidic pH. By contrast, the off-pathway state accumulating transiently during refolding has a higher FRET efficiency, indicating non-native interactions that reduce the Cys10-Trp41 spatial distance, revealing a third distinct conformational state. Overall, our results clarify a negligible degree of unfolding of β-strand C in the formation of the amyloidogenic state and establish the concept that TTR is a highly plastic protein able to populate at least three distinct conformational states.

The acylphosphatase from Sulfolobus solfataricus (Sso AcP) is a globular protein able to aggregate in vitro from a native-like conformational ensemble without the need for a transition across the major unfolding energy barrier. This process leads to the formation of assemblies in which the protein retains its native-like structure, which subsequently convert into amyloid-like aggregates. Here, we investigate the mechanism by which Sso AcP aggregates in vivo to form bacterial inclusion bodies after expression in E. coli. Shortly after the initiation of expression, Sso AcP is incorporated into inclusion bodies as a native-like protein, still exhibiting small but significant enzymatic activity. Additional experiments revealed that this overall process of aggregation is enhanced by the presence of the unfolded N-terminal region of the sequence and by destabilization of the globular segment of the protein. At later times, the Sso AcP molecules in the inclusion bodies lose their native-like properties and convert into β-sheet-rich amyloid-like structures, as indicated by their ability to bind thioflavin T and Congo red. These results show that the aggregation behavior of this protein is similar in vivo to that observed in vitro, and that, at least for a predominant part of the protein population, the transition from a native to an amyloid-like structure occurs within the aggregate state., (Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cerato-platanin (CP) is a non-catalytic, cysteine-rich protein, the first member of the cerato-platanin family. It is a single-domain protein with a double Ψ/β barrel domain resembling the D1 domain of plant and bacterial expansins. Similarly to expansins, CP shows a cell wall-loosening activity on cellulose and can be defined as an expanisin-like protein, in spite of the missing D2 domain, normally present in plant expansins. The weakening activity shown on cellulose may facilitate the CP-host interaction, corroborating the role of CP in eliciting plant defence response. Indeed, CP is an elicitor of primary defences acting as a Pathogen-Associated Molecular Patterns (PAMP). So far, structure-function relationship study has been mainly performed on the bacterial BsEXLX1 expansin, probably due to difficulties in expressing plant expansins in heterologous systems. Here, we report a subcloning and purification method of CP in the engineered E. coli SHuffle cells, which proved to be suitable to obtain the properly folded and biologically active protein. The method also enabled the production of the mutant D77A, rationally designed to be inactive. The wild-type and the mutated CP were characterized for cellulose weakening activity and for PAMP activity (i.e. induction of Reactive Oxygen Species synthesis and phytoalexins production). Our analysis reveals that the carboxyl group of D77 is crucial for expansin-like and PAMP activities, thus permitting to establish a correlation between the ability to weaken cellulose and the capacity to induce defence responses in plants. Our results enable the structural and functional characterization of a mono-domain eukaryotic expansin and identify the essential role of a specific aspartic residue in cellulose weakening.

Folding of polypeptide chains into biologically active entities is an astonishingly complex process, determined by the nature and the sequence of residues emerging from ribosomes. While it has been long believed that evolution has pressed genomes so that specific sequences could adopt unique, functional three-dimensional folds, it is now clear that complex protein machineries act as quality control system and supervise folding. Notwithstanding that, events such as erroneous folding, partial folding, or misfolding are frequent during the life of a cell or a whole organism, and they can escape controls. One of the possible outcomes of this misbehavior is cross-β aggregation, a super secondary structure which represents the hallmark of self-assembled, well organized, and extremely ordered structures termed amyloid fibrils. What if evolution would have not taken into account such possibilities? Twenty years of research point toward the idea that, in fact, evolution has constantly supervised the risk of errors and minimized their impact. In this review we tried to survey the major findings in the amyloid field, trying to describe what the real pitfalls of protein folding are-from an evolutionary perspective-and how sequence and structural features have evolved to balance the need for perfect, dynamic, functionally efficient structures, and the detrimental effects implicit in the dangerous process of folding. We will discuss how the knowledge obtained from these studies has been employed to produce computational methods able to assess, predict, and discriminate the aggregation properties of protein sequences., (© 2017 Elsevier Inc. All rights reserved.)

The first genetic variant of β2 -microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild-type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1-enriched domains only, thus establishing a link between aggregate-membrane contact and cell damage., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The PFN1 gene, coding for profilin-1, has recently been associated with familial amyotrophic lateral sclerosis (fALS), as three mutations, namely C71G, M114T, and G118V, have been found in patients with familial forms of the disease and another, E117G, has been proposed to be a moderate risk factor for disease onset. In this work, we have purified the four profilin-1 variants along with the wild-type protein. The resulting aggregates appear to be fibrillar, to have a weak binding to ThT, and to possess a significant amount of intermolecular β-sheet structure. Using ThT fluorescence assays, far-UV circular dichroism, and dynamic light scattering, we found that all four variants have an aggregation propensity higher than that of the wild-type counterpart. In particular, the C71G mutation was found to induce the most dramatic change in aggregation, followed by the G118V and M114T substitutions and then the E117G mutation. Such a propensity was found not to strictly correlate with the conformational stability in this group of profilin-1 variants, determined using both urea-induced denaturation at equilibrium and folding/unfolding kinetics. However, it correlated with structural changes of the folded states, as monitored with far-UV circular dichroism, intrinsic fluorescence spectroscopy, ANS binding, acrylamide quenching, and dynamic light scattering. Overall, the results suggest that all four mutations increase the tendency of profilin-1 to aggregate and that such aggregation behavior is largely determined by the mutation-induced structural changes occurring in the folded state of the protein.

Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states.

Lymphocyte function-associated antigen 1 (LFA-1) is an integrin that transmits information in two directions across the plasma membrane of leukocytes, in so-called outside-in and inside-out signaling mechanisms. To investigate the structural basis of these mechanisms, we studied the conformational space of the apo I-domain using replica-averaged metadynamics simulations in combination with nuclear magnetic resonance chemical shifts. We thus obtained a free energy landscape that reveals the existence of three conformational substates of this domain. The three substates include conformations similar to existing crystallographic structures of the low-affinity I-domain, the inactive I-domain with an allosteric antagonist inhibitor bound underneath α helix 7, and an intermediate affinity state of the I-domain. The multiple substates were validated with residual dipolar coupling measurements. These results suggest that the presence of three substates in the apo I-domain enables the precise regulation of the binding process that is essential for the physiological function of LFA-1., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lymphocyte function-associated antigen-1 (LFA-1) is an integrin protein that transmits information across the plasma membrane through the so-called inside-out and outside-in signaling mechanisms. To investigate these mechanisms, we carried out an NMR analysis of the dynamics of the LFA-1 I-domain, which has enabled us to characterize the motions of this domain on a broad range of timescales. We studied first the internal motions on the nanosecond timescale by spin relaxation measurements and model-free analysis. We then extended this analysis to the millisecond timescale motions by measuring (15) N-(1) H residual dipolar couplings of the backbone amide groups. We analyzed these results in the context of the three major conformational states of the I-domain using their corresponding X-ray crystallographic structures. Our results highlight the importance of the low-frequency motions of the LFA-1 I-domain in the inactive apo-state. We found in particular that α-helix 7 is in a position in the apo-closed state that cannot be fully described by any of the existing X-ray structures, as it appears to be in dynamic exchange between different conformations. This type of motion seems to represent an inherent property of the LFA-1 I-domain and might be relevant for controlling the access to the allosteric binding pocket, as well as for the downward displacement of α-helix 7 that is required for the activation of LFA-1., (© 2014 The Protein Society.)

β-proteins are constantly threatened by the risk of aggregation because β-sheets are inherently structured for edge-to-edge interactions. To avoid native-like aggregation, evolution has resulted in a set of strategies that prevent intermolecular β-interactions. Acylphosphatase from Sulfolobus solfataricus (Sso AcP) represents a suitable model for the study of such a process. Under conditions promoting aggregation, Sso AcP acquires a native-like conformational state whereby an unstructured N-terminal segment interacts with the edge β-strand B4 of an adjacent Sso AcP molecule. Because B4 is poorly protected against aggregation, this interaction triggers the aggregation cascade without the need for unfolding. Recently, three single Sso AcP mutants (V84D, Y86E and V84P) were designed to engineer additional protection against aggregation in B4 and were observed to successfully impair native-like aggregation in all three variants at the expense of a lower stability. To understand the structural basis of the reduced aggregation propensity and lower stability, the crystal structures of the Sso AcP variants were determined in the present study. Structural analysis reveals that the V84D and Y86E mutations exert protection by the insertion of an edge negative charge. A conformationally less regular B4 underlies protection against aggregation in the V84P mutant. The thermodynamic basis of instability is discussed. Moreover, kinetic experiments indicate that aggregation of the three mutants is not native-like and is independent of the interaction between B4 and the unstructured N-terminal segment. The reported data rationalize previous evidence regarding Sso AcP native-like aggregation and provide a basis for the design of aggregation-free proteins., Database: The atomic coordinates and related experimental data for the Sso AcP mutants V84P, V84D, ΔN11 Y86E have been deposited in the Protein Data Bank under accession numbers 4OJ3, 4OJG and 4OJH, respectively., Structured Digital Abstract: • Sso AcP and Sso AcP bind by fluorescence technology (View interaction)., (© 2014 FEBS.)

Aggregation of transthyretin (TTR) is known to be linked to the development of systemic and localized amyloidoses. It also appears that TTR exerts a protective role against aggregation of the Aβ peptide, a process linked to Alzheimer's disease. In vitro, both processes correlate with the ability of TTR to populate a monomeric state, yet a complete description of the possible conformational states populated by monomeric TTR in vitro at physiological pH is missing. Using an array of biophysical methods and kinetic tests, we show that once monomers of transthyretin are released from the tetramer, equilibrium is established between a set of conformational states possessing different degrees of disorder. A molten globular state appears in equilibrium with the fully folded monomer, whereas an off-pathway species accumulates transiently during refolding of TTR. These two conformational ensembles are distinct in terms of structure, kinetics, and their pathways of formation. Further subpopulations of the protein fold differently because of the occurrence of proline isomerism. The identification of conformational states unrevealed in previous studies opens the way for further characterization of the amyloidogenicity of TTR and its protective role in Alzheimer's disease.

The conversion of normally folded proteins into amyloid-like fibrils is an important process in protein chemistry, biology, pathology and biotechnology. This process generally requires harsh conditions, such as pH extremes, organic cosolvents, high temperatures, high pressures or shear forces. Such conditions promote aggregation because they partially unfold structured proteins or allow the sampling of locally unfolded native-like states, both of which possibly represent amyloidogenic states. Here we report the formation of amyloid-like fibrils by the lipase from Pseudomonas sp. under conditions that are close to physiological, that is, in the absence of denaturants and agitation. The resulting aggregates bind thioflavin T and Congo red, causing their characteristic spectral changes observed in the presence of amyloid fibrils. They possess a significant quantity of β-sheet structure, as detected with Fourier transform infrared and far-UV circular dichroism spectroscopies, and appear fibrillar using transmission electron microscopy. These results indicate that the lipase from Pseudomonas sp. can be a useful model system for the characterization of a key process, such as amyloid fibril formation under physiological conditions.

Folded proteins can access aggregation-prone states without the need for transitions that cross the energy barriers for unfolding. In this study we characterized the initial steps of aggregation from a native-like state of the acylphosphatase from Sulfolobus solfataricus (Sso AcP). Using computer simulations restrained by experimental hydrogen/deuterium (H/D) exchange data, we provide direct evidence that under aggregation-promoting conditions Sso AcP populates a conformational ensemble in which native-like structure is retained throughout the sequence in the absence of local unfolding (N∗), although the protein exhibits an increase in hydrodynamic radius and dynamics. This transition leads an edge strand to experience an increased affinity for a specific unfolded segment of the protein. Direct measurements by means of H/D exchange rates, isothermal titration calorimetry, and intermolecular relaxation enhancements show that after formation of N∗, an intermolecular interaction with an antiparallel arrangement is established between the edge strand and the unfolded segment of the protein. However, under conditions that favor the fully native state of Sso AcP, such an interaction is not established. Thus, these results reveal a novel (to our knowledge) self-assembly mechanism for a folded protein that is based on the increased flexibility of highly aggregation-prone segments in the absence of local unfolding., (Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.

Human muscle acylphosphatase (mAcP) is an enzyme with a ferrodoxin-like topology whose primary role is to hydrolyze the carboxyl-phosphate bonds of acylphosphates. The protein has been widely used as a model system for elucidating the molecular determinants of protein folding and misfolding. We present here the full NMR assignments of the backbone and side chains resonances of mAcP complexed with phosphate, thus providing an important resource for future solution-state NMR spectroscopic studies of the structure and dynamics of this protein in the contexts of protein folding and misfolding.

The conversion of proteins from their native state to misfolded oligomers is associated with, and thought to be the cause of, a number of human diseases, including Alzheimer's disease, Parkinson's disease, and systemic amyloidoses. The study of the structure, mechanism of formation, and biological activity of protein misfolded oligomers has been challenged by the metastability, transient formation, and structural heterogeneity of such species. In spite of these difficulties, in the past few years, many experimental approaches have emerged that enable the detection and the detailed molecular study of misfolded oligomers. In this review, we describe the basic and generic knowledge achieved on protein oligomers, describing the mechanisms of oligomer formation, the methodologies used thus far for their structural determination, and the structural elements responsible for their toxicity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

It has previously been shown that the acylphosphatase from Sulfolobus solfataricus is capable of forming amyloid-like aggregates under conditions in which the native structure is maintained and via the transient formation of native-like aggregates. Based on the previously determined NMR structure of the native protein, showing a ferredoxin-like fold and the peculiar presence of an unstructured N-terminal segment, we show here, at a molecular level using NMR spectroscopy, that indeed S. solfataricus acylphosphatase remains in a native-like conformation when placed in aggregating conditions and that such a native-like structure persists when the protein forms the initial aggregates, at least within the low molecular weight species. The analysis carried out under different solution conditions, based on the measurement of the combined (1)H and (15)N chemical shifts and hydrogen/deuterium exchange rates, enabled the most significant conformational changes to be monitored upon transfer of the monomeric state into aggregating conditions and upon formation of the initial native-like aggregates. Important increases of the hydrogen/deuterium exchange rates throughout the native protein, accompanied by small and localized structural changes, in the monomeric protein were observed. The results also allow the identification of the intermolecular interaction regions within the native-like aggregates, that involve, in particular, the N-terminal unstructured segment, the apical region including strands S4 and S5 with the connecting loop, and the opposite active site.

Studies in vitro show that globular proteins can experience the formation of native-like conformational states able to self-assemble with no need of transitions across the energy barrier for unfolding, and that such processes can lead eventually to the formation of amyloid-like species. Circumstantial evidence collected in vivo suggests that aggregation of native-like states can be a concrete possibility for living organisms and thus more relevant than previously thought. In this review we summarize the key observations collected on the "native-like aggregation" of the acylphosphatase from Sulfolobus solfataricus, a protein that has allowed the direct monitoring and analysis of native-like aggregates for its propensity to form rapidly native-like aggregates and their slow conversion into amyloid-like aggregates.

Evidence is accumulating that normally folded proteins retain a significant tendency to form amyloid fibrils through a direct assembly of monomers in their native-like conformation. However, the factors promoting such processes are not yet well understood. The acylphosphatase from Sulfolobus solfataricus (Sso AcP) aggregates under conditions in which a native-like state is initially populated and forms, as a first step, aggregates in which the monomers maintain their native-like topology. An unstructured N-terminal segment and an edge beta-strand were previously shown to play a major role in the process. Using kinetic experiments on a set of Sso AcP variants we shall show that the major event of the first step is the establishment of an inter-molecular interaction between the unstructured segment of one Sso AcP molecule and the globular unit of another molecule. This interaction is determined by the primary sequence of the unstructured segment and not by its physico-chemical properties. Moreover, we shall show that the conversion of these initial aggregates into amyloid-like protofibrils is an intra-molecular process in which the Sso AcP molecules undergo conformational modifications. The obtained results allow the formulation of a model for the assembly of Sso AcP into amyloid-like aggregates at a molecular level.

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k(F)(H2O)=72,600 s(-1)), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.

As structural flexibility is known to be required for enzyme catalysis and pattern recognition and a significant fraction of eukaryotic proteins appear to be unfolded or contain unstructured regions, biological activity of conformational states distinct from fully folded structures could be more common than previously thought. By applying a procedure that allows the recovery of enzymatic activity to be monitored in real time, we show that a non-native state populated transiently during folding of the acylphosphatase from Sulfolobus solfataricus is enzymatically active. The structural characterization of this partially folded state reveals that enzymatic activity is possible even if the catalytic site is structurally heterogeneous, whereas the remainder of the structure acts as a scaffold. These results extend the spectrum of biological functions carried out in the absence of a folded state to include enzyme catalysis.

The assembly of proteins into highly organized fibrillar aggregates is a key process in biology, biotechnology, and human disease. It has been shown that proteins retain a small, yet significant propensity to aggregate when they are folded into compact globular structures, and this may be physiologically relevant, particularly when considering that proteins spend most of their lifespan into such compact states. Proteins from the acylphosphatase-like structural family have been shown to aggregate via different mechanisms, with some members forming native-like aggregates as a first step of their aggregation process and others requiring unfolding as a first necessary step. Here we use the acylphosphatase from Sulfolobus solfataricus to show that assembly of folded protein molecules into native-like aggregates is prevented by single-point mutations that introduce structural protections within one of the most flexible region of the protein, the peripheral edge beta-strand 4. The resulting mutants do not form native-like aggregates, but can still form thioflavin T-binding and beta-structured oligomers, albeit more slowly than the wild-type protein. The kinetic data show that formation of the latter species proceeds via an alternative mechanism that is independent of the transient formation of native-like aggregates.

Amyloid fibril formation is a process that represents an essential feature of the chemistry of proteins and plays a central role in human pathology and the biology of living organisms. In this Account, we shall describe some of the recent results on the sequence and structural determinants of protein aggregation. We shall describe the factors that govern aggregation of unfolded peptides and proteins. We shall then try to summarize the factors that pertain to the aggregation of partially structured states and will show that even fully folded states of proteins have an ability to aggregate into at least early oligomers with no need to undergo substantial conformational changes.