Oligomerization Profile of Human Transthyretin Variants with Distinct Amyloidogenicity

One of the molecular hallmarks of amyloidoses is ordered protein aggregation involving the initial formation of soluble protein oligomers that eventually grow into insoluble fibrils. The identification and characterization of molecular species critical for amyloid fibril formation and disease development have been the focus of intense analysis in the literature. Here, using photo-induced cross-linking of unmodified proteins (PICUP), we studied the early stages of oligomerization of human transthyretin (TTR), a plasma protein involved in amyloid diseases (ATTR amyloidosis) with multiple clinical manifestations. Upon comparison, the oligomerization processes of wild-type TTR (TTRwt) and several TTR variants (TTRV30M, TTRL55P, and TTRT119M) clearly show distinct oligomerization kinetics for the amyloidogenic variants but a similar oligomerization mechanism. The oligomerization kinetics of the TTR amyloidogenic variants under analysis showed a good correlation with their amyloidogenic potential, with the most amyloidogenic variants aggregating faster (TTRL55P > TTRV30M > TTRwt). Moreover, the early stage oligomerization mechanism for these variants involves stepwise addition of monomeric units to the growing oligomer. A completely different behavior was observed for the nonamyloidogenic TTRT119M variant, which does not form oligomers in the same acidic conditions and even for longer incubation times. Thorough characterization of the initial steps of TTR oligomerization is critical for better understanding the origin of ATTR cytotoxicity and developing novel therapeutic strategies for the treatment of ATTR amyloidosis.     

Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry

The precise mechanism of protein folding remains elusive and there is a deficiency of biophysical techniques that are capable of monitoring the individual behavior of copopulated protein conformers during the folding process. Herein, an ion mobility spectrometry (IMS) device integrated with electrospray ionization mass spectrometry (ESI-MS) has been used to successfully separate and analyze protein conformers differing in cross section and/or charge state. In an initial test, an ensemble of folded and partially folded conformers of the protein cytochrome c was separated. A detailed study undertaken on the amyloidogenic protein beta(2)-microglobulin (beta(2)m), which forms fibrils by protein unfolding followed by self-aggregation and is responsible for the disease dialysis-related amyloidosis, has generated important insights into its folding landscape. Initially, a systematic titration of beta(2)m over the pH range 2 to 7 using ESI-IMS-MS allowed individual conformers to be monitored and quantified throughout the acid denaturation process. Furthermore, a comparison of wild-type beta(2)m with single and double amino acid variants with a range of folding stabilities and propensities for amyloid fibril formation has provided illuminating evidence of the role of different conformers in protein stability and amyloidogenic aggregation. The ESI-IMS-MS data presented here not only demonstrate an important and informative further dimension to ESI-MS, but also illustrate the potential of the ESI-IMS-MS technique for unravelling protein folding enigmas in general and studying protein misfolding diseases in particular.     

Molecular mechanisms of polypeptide aggregation in human diseases

Protein aggregation is implicated in a plethora of neurodegenerative diseases. The proteins found to aggregate in these diseases are unrelated in their native structures and amino acid sequences, but form similar insoluble fibrils with characteristic cross-beta sheet morphologies called amyloid in the aggregated state. While both the mechanism of aggregation and the structure of the aggregates are not fully understood at the molecular level, recent studies provide strong support for the idea that protein aggregation into highly stable, insoluble amyloid structures is a general property of the polypeptide chain. For proteins with a unique native state, it is known that aggregation occurs under conditions that promote native-state destabilization in vitro and in vivo. Taken together, the results of several important recent investigations suggest three broad molecular frameworks that may underlie the conversion of normally soluble peptides and proteins into insoluble amyloid fibrils: (1) edge-strand hydrogen bonding, (2) domain-swapping, and (3) self-association of amyloidogenic fragments. We argue that these underlying scenarios are not mutually exclusive and may be protein-dependent - i.e., a protein with a high content of hinge-regions may aggregate via a runaway domain-swap, whereas a protein with a high content of amyloidogenic fragments may aggregate primarily by the self-association of these fragments. These different scenarios provide frameworks to understand the molecular mechanism of polypeptide aggregation.     

Peptide plane can flip in two opposite directions: implication in amyloid formation of transthyretin

Transthyretin (TTR) is one of the known 20 or so human proteins that form fibrils in vivo, which is a hallmark of amyloid diseases. Recently, molecular dynamics simulations using ENCAD force field have revealed that under low pH conditions, the peptide planes of several amyloidogenic proteins can flip in one direction to form an alpha-pleated structure which may be a common conformational transition in the fibril formation. We performed molecular dynamics simulations with AMBER force fields on a recently engineered double mutant TTR, which was shown experimentally to form amyloid fibrils even under close to physiological conditions. Our simulations have demonstrated that peptide-plane flipping can occur even under neutral pH and room temperature for this amyloidogenic TTR variant. Unlike previously reported peptide-plane flipping of TTR using ENCAD force field, we have found two-way flipping using AMBER force field. We propose a new mechanism of amyloid formation based on the two-way flipping, which gives a better explanation of various experimental and computational results. In principle, the residual dipolar and hydrogen-bond scalar coupling techniques can be applied to the wild-type TTR and the variant to study the peptide-plane flipping of amyloidogenic proteins.     

Population of nonnative states of lysozyme variants drives amyloid fibril formation

The propensity of protein molecules to self-assemble into highly ordered, fibrillar aggregates lies at the heart of understanding many disorders ranging from Alzheimer's disease to systemic lysozyme amyloidosis. In this paper we use highly accurate kinetic measurements of amyloid fibril growth in combination with spectroscopic tools to quantify the effect of modifications in solution conditions and in the amino acid sequence of human lysozyme on its propensity to form amyloid fibrils under acidic conditions. We elucidate and quantify the correlation between the rate of amyloid growth and the population of nonnative states, and we show that changes in amyloidogenicity are almost entirely due to alterations in the stability of the native state, while other regions of the global free-energy surface remain largely unmodified. These results provide insight into the complex dynamics of a macromolecule on a multidimensional energy landscape and point the way for a better understanding of amyloid diseases.     

Cytochrome display on amyloid fibrils

Protein amyloid fibrils can be functionalized by coating the core protofilament with high concentrations of proteins and enzymes. This can be done elegantly by appending a functional domain to an amyloidogenic protein monomer, then assembling the monomers into a fibril. To display an array of biologically functional porphyrins on the surface of protein fibrils, we have fused the sequence of the small, soluble cytochrome b562 to an SH3 dimer sequence that can form classical amyloid fibrils rapidly under well-defined conditions. The resulting fusion protein also forms amyloid fibrils and, in addition, binds metalloporphyrins, at half of the porphyrin binding sites as shown by UV-vis and NMR spectroscopies. Once metalloporphyrins are bound to the fibrils, the resulting holo-cytochrome domains are spectroscopically identical to the wild type cytochrome. The concentration of metalloporphyrins on a saturated fibril is estimated to be of the order of approximately 20 mM, suggesting that they could be interesting systems for applications in nanotechnology.     

Unfolding and refolding details of lysozyme in the presence of β-casein micelles

In this work, we selected a small globular protein, lysozyme, to study how it unfolds and refolds in the presence of micelles composed of the unstructured β-casein proteins by using microcalorimetry and circular dichroism spectroscopy. It was found that a partially unfolded structure of lysozyme starts to form when the β-casein/lysozyme molar ratio is above 0.7, and the structure forms exclusively when the β-casein/lysozyme molar ratio is above 1.6. This partially unfolded state of lysozyme loses most of its tertiary structure and after heating, the denatured lysozyme molecules are trapped in the charged coatings of β-casein micelles and cannot refold upon cooling. The thus obtained protein complex can be viewed as a kind of special polyelectrolyte complex micelle. The net charge ratios of the two proteins and the ionic strength of the dispersions can significantly modulate the electrostatic and hydrophobic interactions between the two proteins. Our present work may have implications for the nanoparticle protein engineering therapy in the biomedicine field and may provide a better understanding of the principles governing the protein-protein interactions. Besides, the heating-cooling-reheating procedure employed in this work can also be used to study the unfolding and refolding details of the target protein in other protein-protein, protein-polymer and protein-small solute systems.     

Capillary electrophoresis investigation of a partially unfolded conformation of beta(2)-microglobulin

Dialysis-related amyloidosis is a disease in which partial unfolding of beta(2)-microglobulin plays a key pathogenetic role in the formation of the amyloid fibrils. We have recently demonstrated that a partially unfolded conformer of beta(2)-microglobulin is involved in fibrillogenesis and that this species is significantly populated under physiological conditions. In this work capillary electrophoresis has been used to measure the equilibrium between the native protein and this conformer in samples known to have a higher or lower amyloidogenic potential, namely full-length beta(2)-microglobulin, two truncated species and a mutant, created by replacing histidine in position 31 with thyrosine. In addition, for all protein species folding stability experiments have been carried out by monitoring the secondary structure by circular dichroism at increasing concentrations of guanidinium chloride. The values of free energy of unfolding in the absence of denaturant, obtained by elaboration of these experiments, were found to be inversely correlated to the area percent of the partially unfolded conformer, as measured by capillary electrophoresis. Affinity capillary electrophoresis experiments have been also carried out under nondenaturing conditions to assess the affinity of copper and suramin to either the native form or the conformational intermediate of full-length beta(2)-microglobulin.     

Amino Acid Functionalized Superparamagnetic Nanoparticles Inhibit Lysozyme Amyloid Fibrillization

Nanoparticles have great potential to be used in various biomedical applications, including therapy or diagnosis of amyloid-related diseases. The physical and chemical properties of iron oxide superparamagnetic nanoparticles (MNPs) functionalized with different amino acids (AAs), namely, with lysine (Lys), glycine (Gly), or tryptophan (Trp), have been characterized. The cytotoxicity of nanoparticles and their effect on amyloid fibrillization of lysozymes in vitro was also verified. The AA-MNPs under study are nontoxic to human SHSY5Y neuroblastoma cells. Moreover, the AA-MNPs were able to significantly inhibit lysozyme amyloid fibrillization and destroy amyloid fibrils. Kinetic studies revealed that the presence of AA-MNPs affected lysozyme fibrillization, namely, the lag phase and steady-state phase of the growth curves. The most effective activities were observed for Trp-MNPs, which revealed the importance of aromatic rings in the structure of AAs used as coating agents. The obtained results indicate the possible application of these AA-MNPs in the treatment of amyloid diseases associated with lysozyme or other amyloidogenic proteins.     

Theoretical investigations of TTR derived aggregation-prone peptides’ potential to biochemically attenuate the amyloidogenic propensities of V30 M TTR amyloid fibrils

Transthyretin (TTR) is a cerebrospinal fluid and plasma prevalent protein implicated in heritable and sporadic amyloidosis. Numerous mutations and a wide range of phenotypes have been associated with TTR-mediated amyloidosis. Among these, V30 M is the most predominant point mutation, inculpated with familial amyloid polyneuropathy (FAP), a life-threatening autosomal dominant genetic disorder characterized by the deposition of amyloid fibrils in crucial areas. Hence, efficacious therapeutics against this detrimental disorder is warranted. Lately, several peptide-based analeptics, especially the ones that are aggregation-prone and the ones derived from aggregation hotspots of amyloidogenic proteins are being increasingly proffered against the amyloid fibrils. In the present study, as an effective precursor to in vitro investigations, we examined and assessed the therapeutic potentials of aggregation-prone peptides (APPs) derived from TTR, against V30 M TTR amyloid fibrils, computationally. Out of five experimentally corroborated APPs availed for this study, molecular dynamics simulation analysis endorses APP TAVVTN to be an effective beta-sheet breaker against V30 M TTR amyloid fibrils. Furthermore, consistent findings from various molecular trajectory analyses, residual frustration analysis and simulated thermal denaturation have indicated that APP TAVVTN could effectually relater the structural dynamics of V30 M TTR amyloid fibrils, to conformationally digress it away from its amyloidogenic propensities. Hence, based on consistent unvarying findings from numerous adept computational pipelines, APP TAVVTN could be an efficacious analeptic to therapeutically intervene and mitigate the amyloidogenic propensities of V30 M TTR amyloid fibrils, thereby ameliorating the pathological ramifications due to FAP.     

SDS-induced multi-stage unfolding of a small globular protein through different denatured states revealed by single-molecule fluorescence

Ionic surfactants such as sodium dodecyl sulfate (SDS) unfold proteins in a much more diverse yet effective way than chemical denaturants such as guanidium chloride (GdmCl). But how these unfolding processes compare on a molecular level is poorly understood. Here, we address this question by scrutinising the unfolding pathway of the globular protein S6 in SDS and GdmCl with single-molecule Förster resonance energy transfer (smFRET) spectroscopy. We show that the unfolding mechanism in SDS is strikingly different and convoluted in comparison to denaturation in GdmCl. In contrast to the reversible two-state unfolding behaviour in GdmCl characterised by kinetics on the timescale of seconds, SDS demonstrated not one, but four distinct regimes of interactions with S6, dependent on the surfactant concentration. At ≤1 mM SDS, S6 and surfactant molecules form quasi-micelles on a minute timescale; at millimolar [SDS], the protein denatures through an unfolded/denatured ensemble of highly heterogeneous states on a multi-second timescale; at tens of millimolar of SDS, the protein unfolds into a micelle-packed conformation on the second timescale; and >50 mM SDS, the protein unfolds with millisecond timescale dynamics. We propose a detailed model for multi-stage unfolding of S6 in SDS, which involves at least three different types of denatured states with different level of compactness and dynamics and a continually changing landscape of interactions between protein and surfactant. Our results highlight the great potential of single-molecule fluorescence as a direct probe of nanoscale protein structure and dynamics in chemically complex surfactant environments.

Multi-stage unfolding of S6 in SDS involving various types of denatured states with different levels of compactness and dynamics.     

Cold Denaturation of α‐Synuclein Amyloid Fibrils

Although amyloid fibrils are associated with numerous pathologies, their conformational stability remains largely unclear. Herein, we probe the thermal stability of various amyloid fibrils. α‐Synuclein fibrils cold‐denatured to monomers at 0–20 °C and heat‐denatured at 60–110 °C. Meanwhile, the fibrils of β2‐microglobulin, Alzheimer’s Aβ1‐40/Aβ1‐42 peptides, and insulin exhibited only heat denaturation, although they showed a decrease in stability at low temperature. A comparison of structural parameters with positive enthalpy and heat capacity changes which showed opposite signs to protein folding suggested that the burial of charged residues in fibril cores contributed to the cold denaturation of α‐synuclein fibrils. We propose that although cold‐denaturation is common to both native proteins and misfolded fibrillar states, the main‐chain dominated amyloid structures may explain amyloid‐specific cold denaturation arising from the unfavorable burial of charged side‐chains in fibril cores.     

Amyloid‐β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect

Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β‐rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer′s disease amyloid‐β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP‐amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases.     

Reverse Engineering Analysis of the High-Temperature Reversible Oligomerization and Amyloidogenicity of PSD95-PDZ3

PSD95-PDZ3, the third PDZ domain of the post-synaptic density-95 protein (MW 11 kDa), undergoes a peculiar three-state thermal denaturation (N ↔ I_n ↔ D) and is amyloidogenic. PSD95-PDZ3 in the intermediate state (I) is reversibly oligomerized (RO: Reversible oligomerization). We previously reported a point mutation (F340A) that inhibits both ROs and amyloidogenesis and constructed the PDZ3-F340A variant. Here, we “reverse engineered” PDZ3-F340A for inducing high-temperature RO and amyloidogenesis. We produced three variants (R309L, E310L, and N326L), where we individually mutated hydrophilic residues exposed at the surface of the monomeric PDZ3-F340A but buried in the tetrameric crystal structure to a hydrophobic leucine. Differential scanning calorimetry indicated that two of the designed variants (PDZ3-F340A/R309L and E310L) denatured according to the two-state model. On the other hand, PDZ3-F340A/N326L denatured according to a three-state model and produced high-temperature ROs. The secondary structures of PDZ3-F340A/N326L and PDZ3-wt in the RO state were unfolded according to circular dichroism and differential scanning calorimetry. Furthermore, PDZ3-F340A/N326L was amyloidogenic as assessed by Thioflavin T fluorescence. Altogether, these results demonstrate that a single amino acid mutation can trigger the formation of high-temperature RO and concurrent amyloidogenesis.     

The formation of nematic liquid crystal phases by hen lysozyme amyloid fibrils

Amyloid fibrils are a polymeric aggregate of protein. The fibrils are typically on the order of micrometers long, with widths of 10-20 nm. They are generally regarded as stiff, and nonbranching. It is well-known that similar synthetic polymers and biopolymers such as DNA and polysaccharides, have a tendency to form liquid crystalline phases when incubated under appropriate conditions. Here we show that amyloid fibrils from the protein hen lysozyme can similarly form liquid crystal phases. The most common phase observed is the nematic. Alignment can persist for several centimeters. When the fibrils are freeze-thawed to shorten them, similar phases form but at higher concentrations, confirming the importance of the aspect ratio of the fibrils. Freeze-thawed fibrils are also seen to form "tactoids", discrete liquid crystalline structures. The addition of NaCl to the solutions appears to only have a minor effect, while the effect of pH appears much more significant. We propose that the consideration of amyloid fibrils as polymer analogues should open new routes to explore in the burgeoning field of biomaterials.     

Measurement of electrostatic interactions in protein folding with the use of protein charge ladders

This paper describes a new method for the measurement of the role of interactions between charged groups on the energetics of protein folding. This method uses capillary electrophoresis (CE) and protein charge ladders (mixtures of protein derivatives that differ incrementally in number of charged groups) to measure, in a single set of electrophoresis experiments, the free energy of unfolding (DeltaG(D-N)) of alpha-lactalbumin (alpha-LA) as a function of net charge. These same data also yield the hydrodynamic radius, R(H), and net charge measured by CE, Z(CE), of the folded and denatured proteins. Alpha-LA unfolds to a compact denatured state under mildly alkaline conditions; a small increase in R(H) (11%, 2 A) coincides with a large increase in Z(CE) (71%, -4 charge units), relative to the folded state. The increase in Z(CE), in turn, predicts a large pH dependence of free energy of unfolding (-22 kJ/mol per unit increase in pH), due to differences in proton binding in the folded and denatured states. The free energy of unfolding correlates with the square of net charge of the members of the charge ladder. The differential dependence of DeltaG(D-N) on net charge for holo-alpha-LA, (partial differential) DeltaG(D-N)/(partial differential)Z = -0.14Z kJ/mol per unit of charge. This dependence of DeltaG(D-N) on net charge is a result of a net electrostatic repulsion among charge groups on the protein. These results, together with data from pH titrations, show that both the effects of electrostatic repulsion and differences in proton binding in the folded and denatured states can play an important role in the pH dependence of this protein; the relative magnitude of these effects varies with pH. The combination of charge ladders and CE is a rapid and efficient tool that measures the contributions of electrostatics to the energetics of protein folding, and the size and charge of proteins as they unfold. All this information is obtained from a single set of electrophoresis experiments.     

Identification of amyloidogenic peptide sequences using a coarse-grained physicochemical model

Cross-beta amyloid is implicated in over 20 human diseases. Experiments suggest that specific sequence elements within amyloidogenic proteins play a major role in seeding amyloid formation. Identifying these seeding sequences is important for rationalizing the molecular mechanisms of amyloid formation and for elaborating therapeutic strategies that target amyloid. Theoretical techniques play an important role in facilitating the identification and structural characterization of putative seeding sequences; most amyloid species are not amenable to high resolution experimental structure techniques. In this study we have combined a coarse-grained physicochemical protein model with a highly efficient Monte Carlo sampling technique to identify amyloidogenic sequences in four proteins for which respective experimental peptide fragmentation data exist. Peptide sequences were defined as amyloidogenic if the ensemble structure predicted for three interacting peptides described a stable and regular three-stranded beta-sheet. For such peptides, free energies were calculated to provide a measure of amyloid propensity. The overall agreement between the experimental and predicted data is good, and we correctly identify several self-recognition motifs proposed to define the cross-beta amyloid fibril architectures of two of the proteins. Our results compare very favorably with those obtained using atomistic molecular dynamics methods, though our simulations are 30-40 times faster.     

The yin and yang of amyloid: insights from α-synuclein and repeat domain of Pmel17

Amyloid has been traditionally viewed in the context of disease. However, the emerging concept of 'functional amyloid' has taken a new direction into how we view amyloid. Recent studies have identified amyloid fibrils ranging from bacteria to humans that have a beneficial role, instead of being associated with a misfolded state that has been implicated in diseases such as Alzheimer's, Parkinson's and prion diseases. Here, we review our work on two human amyloidogenic polypeptides, one associated with Parkinson's disease, α-synuclein (α-syn), and the other important for melanin synthesis, the repeat domain (RPT) from Pmel17. Particularly, we focused our attention on spectroscopic studies of protein conformation and dynamics and their impact on α-syn amyloid formation and for RPT, we discussed the strict pH dependence of amyloid formation and its role in melanin biosynthesis.     

End Capping Alters the Structural Characteristics and Mechanical Properties of Transthyretin (105–115) Amyloid Protofibrils

Pathological amyloid proteins are associated with degenerative and neurodegenerative diseases. These amyloid proteins develop as oligomer, fibrillar, and plaque forms, due to the denatured and unstable status of the amyloid monomers. Specifically, the development of fibrillar amyloid proteins has been investigated through several experimental studies. To understand the generation of amyloid fibrils, environmental factors such as point mutations, pH, and polymorphic characteristics have been considered. Recently, amyloid fibril studies related to end‐capping effects have been conducted to understand amyloid fibril development. However, atomic‐level studies to determine the stability and mechanical properties of amyloid fibrils based on end capping have not been undertaken. In this study, we show that end capping alters the structural characteristics and conformations of transthyretin (TTR) amyloid fibrils by using molecular dynamics (MD) simulations. Variation in the structural conformations and characteristics of the TTR fibrils through end capping are observed, due to the resulting electrostatic energies and hydrophobicity characteristics. Moreover, the end capping changes the mechanical properties of TTR fibrils. Our results shed light on amyloid fibril formation under end‐capping conditions.     

Self-assembly of short amyloidogenic peptides at the air-water interface

Short peptide stretches in amyloidogenic proteins can form amyloid fibrils in vitro and have served as good models for studying amyloid fibril formation. Recently, these amyloidogenic peptides have gained considerable attention, as non-amyloid ordered structures can be obtained from these peptides by carefully tuning the conditions of self-assembly, especially pH, temperature and presence of organic solvents. We have examined the effect of surface pressure on the self-assembled structures of two amyloidogenic peptides, Pβ(2)m (Ac-DWSFYLLYYTEFT-am) and AcPHF6 (Ac-VQIVYK-am) at the air-water interface when deposited from different solvents. Both the peptides are surface-active and form Thioflavin T (ThT) positive structures at the air-water interface. There is considerable hysteresis in the compression and expansion isotherms, suggesting the occurrence of structural rearrangements during compression. Preformed Pβ(2)m fibrillar structures at the air-water interface are disrupted as peptide is compressed to lower molecular areas but restored if the film is expanded, suggesting that the process is reversible. AcPHF6, on the other hand, shows largely sheet-like structures at lower molecular areas. The solvents used for dissolution of the peptides appear to influence the nature of the aggregates formed. Our results show that like hydrostatic pressure, surface pressure can also be utilized for modulating the self-assembly of the amyloidogenic and self-assembling peptides.