Using Capillary Electrophoresis to Investigate Protein Conformational and Compositional Heterogeneity.

Detailed insights into protein structure/function relationships require robust characterization methodologies. Free-solution capillary electrophoresis (CE) is a unique separation technique which is sensitive to the conformation and/or composition of proteins, and therefore provides information on the heterogeneity of these properties. Three unrelated, conformationally/compositionally-altered proteins were separated by CE. An electrophoretic mobility distribution was determined for each protein along with its conformational and/or compositional heterogeneity. The CE results were compared with molar mass distributions obtained from size-exclusion chromatography coupled to light scattering (SEC-MALS). Bovine serum albumin multimers and two monomeric species were separated, highlighting variations in conformational/compositional heterogeneity among the multimers. Analysis of yeast alcohol dehydrogenase resolved two monomeric conformers and various tetrameric species, illustrating the impact of zinc ion removal and disulfide bond reduction on the protein's heterogeneity. The apo (calcium-free) and holo forms of bovine α-lactalbumin were separated and differences in the species' heterogeneity were measured; by contrast, the SEC-MALS profiles were identical. Comparative analysis of these structurally unrelated proteins provided novel insights into the interplay between molar mass and conformational/compositional heterogeneity. Overall, this study expands the utility of CE by demonstrating its capacity to discern protein species and their heterogeneity, properties which are not readily accessible by other analytical techniques.

The molecular chaperone ß-casein prevents amorphous and fibrillar aggregation of α-lactalbumin by stabilisation of dynamic disorder.

Deficits in protein homeostasis (proteostasis) are typified by the partial unfolding or misfolding of native proteins leading to amorphous or fibrillar aggregation, events that have been closely associated with diseases including Alzheimer's and Parkinson's diseases. Molecular chaperones are intimately involved in maintaining proteostasis, and their mechanisms of action are in part dependent on the morphology of aggregation-prone proteins. This study utilised native ion mobility-mass spectrometry to provide molecular insights into the conformational properties and dynamics of a model protein, α-lactalbumin (α-LA), which aggregates in an amorphous or amyloid fibrillar manner controlled by appropriate selection of experimental conditions. The molecular chaperone ß-casein (ß-CN) is effective at inhibiting amorphous and fibrillar aggregation of α-LA at sub-stoichiometric ratios, with greater efficiency against fibril formation. Analytical size-exclusion chromatography demonstrates the interaction between ß-CN and amorphously aggregating α-LA is stable, forming a soluble high molecular weight complex, whilst with fibril-forming α-LA the interaction is transient. Moreover, ion mobility-mass spectrometry (IM-MS) coupled with collision-induced unfolding (CIU) revealed that α-LA monomers undergo distinct conformational transitions during the initial stages of amorphous (order to disorder) and fibrillar (disorder to order) aggregation. The structural heterogeneity of monomeric α-LA during fibrillation is reduced in the presence of ß-CN along with an enhancement in stability, which provides a potential means for preventing fibril formation. Together, this study demonstrates how IM-MS and CIU can investigate the unfolding of proteins as well as examine transient and dynamic protein-chaperone interactions, and thereby provides detailed insight into the mechanism of chaperone action and proteostasis mechanisms.

The Effect of Oxidized Dopamine on the Structure and Molecular Chaperone Function of the Small Heat-Shock Proteins, αB-Crystallin and Hsp27.

Oxidation of the neurotransmitter, dopamine (DA), is a pathological hallmark of Parkinson's disease (PD). Oxidized DA forms adducts with proteins which can alter their functionality. αB-crystallin and Hsp27 are intracellular, small heat-shock molecular chaperone proteins (sHsps) which form the first line of defense to prevent protein aggregation under conditions of cellular stress. In vitro, the effects of oxidized DA on the structure and function of αB-crystallin and Hsp27 were investigated. Oxidized DA promoted the cross-linking of αB-crystallin and Hsp27 to form well-defined dimer, trimer, tetramer, etc., species, as monitored by SDS-PAGE. Lysine residues were involved in the cross-links. The secondary structure of the sHsps was not altered significantly upon cross-linking with oxidized DA but their oligomeric size was increased. When modified with a molar equivalent of DA, sHsp chaperone functionality was largely retained in preventing both amorphous and amyloid fibrillar aggregation, including fibril formation of mutant (A53T) α-synuclein, a protein whose aggregation is associated with autosomal PD. In the main, higher levels of sHsp modification with DA led to a reduction in chaperone effectiveness. In vivo, DA is sequestered into acidic vesicles to prevent its oxidation and, intracellularly, oxidation is minimized by mM levels of the antioxidant, glutathione. In vitro, acidic pH and glutathione prevented the formation of oxidized DA-induced cross-linking of the sHsps. Oxidized DA-modified αB-crystallin and Hsp27 were not cytotoxic. In a cellular context, retention of significant chaperone functionality by mildly oxidized DA-modified sHsps would contribute to proteostasis by preventing protein aggregation (particularly of α-synuclein) that is associated with PD.

The Amyloid Fibril-Forming ß-Sheet Regions of Amyloid ß and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

A Spectroscopic Marker for Structural Transitions Associated with Amyloid-ß Aggregation.

An amyloid aggregate evolves through a series of intermediates that have different secondary structures and intra- and intermolecular contacts. The structural parameters of these intermediates are important determinants of their toxicity. For example, the early oligomeric species of the amyloid-ß (Aß) peptide have been implicated as the most cytotoxic species in Alzheimer's disease but are difficult to identify because of their dynamic and transitory nature. Conventional aggregation monitors such as the fluorescent dye thioflavin T report on only the overall transition of the soluble species to the final amyloid fibrillar aggregated state. Here, we show that the fluorescent dye bis(triphenylphosphonium) tetraphenylethene (TPE-TPP) identifies at least three distinct aggregation intermediates of Aß. Some atomic-level features of these intermediates are known from solid state nuclear magnetic resonance spectroscopy. Hence, the TPE-TPP fluorescence data may be interpreted in terms of these Aß structural transitions. Steady state fluorescence and lifetime characteristics of TPE-TPP distinguish between the small oligomeric species (emission wavelength maximum, λmax = 465 nm; average fluorescence lifetime, τFl measured at 420 nm = 3.58 ± 0.04 ns), the intermediate species (λmax = 452 nm; τFl = 3.00 ± 0.03 ns), and the fibrils (λmax = 406 nm; τFl = 5.19 ± 0.08 ns). Thus, TPE-TPP provides a ready diagnostic for differentiating between the various, including the toxic, Aß aggregates and potentially can be utilized to screen for amyloid aggregation inhibitors.

The Kinetics of Amyloid Fibrillar Aggregation of Uperin 3.5 Is Directed by the Peptide's Secondary Structure.

Many peptides aggregate into insoluble ß-sheet rich amyloid fibrils. Some of these aggregation processes are linked to age-related diseases, such as Alzheimer's disease and type 2 diabetes. Here, we show that the secondary structure of the peptide uperin 3.5 directs the kinetics and mechanism of amyloid fibrillar aggregation. Uperin 3.5 variants were investigated using thioflavin T fluorescence assays, circular dichroism spectroscopy, and structure prediction methods. Our results suggest that those peptide variants with a strong propensity to form an α-helical secondary structure under physiological conditions are more likely to aggregate into amyloid fibrils than peptides in an unstructured or "random coil" conformation. This conclusion is in good agreement with the hypothesis that an α-helical transition state is required for peptide aggregation into amyloid fibrils. Specifically, uperin 3.5 variants in which charged amino acids were replaced by alanine were richer in α-helical content, leading to enhanced aggregation compared to that of wild type uperin 3.5. However, the addition of 2,2,2-trifluoroethanol as a major co-solute or membrane-mimicking phospholipid environments locked uperin 3.5 to the α-helical conformation preventing amyloid aggregation. Strategies for stabilizing peptides into their α-helical conformation could provide therapeutic approaches for overcoming peptide aggregation-related diseases. The impact of the physiological environment on peptide secondary structure could explain aggregation processes in a cellular environment.

Role of salt bridges in the dimer interface of 14-3-3ζ in dimer dynamics, N-terminal α-helical order, and molecular chaperone activity.

The 14-3-3 family of intracellular proteins are dimeric, multifunctional adaptor proteins that bind to and regulate the activities of many important signaling proteins. The subunits within 14-3-3 dimers are predicted to be stabilized by salt bridges that are largely conserved across the 14-3-3 protein family and allow the different isoforms to form heterodimers. Here, we have examined the contributions of conserved salt-bridging residues in stabilizing the dimeric state of 14-3-3ζ. Using analytical ultracentrifugation, our results revealed that Asp21 and Glu89 both play key roles in dimer dynamics and contribute to dimer stability. Furthermore, hydrogen-deuterium exchange coupled with mass spectrometry showed that mutation of Asp21 promoted disorder in the N-terminal helices of 14-3-3ζ, suggesting that this residue plays an important role in maintaining structure across the dimer interface. Intriguingly, a D21N 14-3-3ζ mutant exhibited enhanced molecular chaperone ability that prevented amorphous protein aggregation, suggesting a potential role for N-terminal disorder in 14-3-3ζ's poorly understood chaperone action. Taken together, these results imply that disorder in the N-terminal helices of 14-3-3ζ is a consequence of the dimer-monomer dynamics and may play a role in conferring chaperone function to 14-3-3ζ protein.

Proteostasis and the Regulation of Intra- and Extracellular Protein Aggregation by ATP-Independent Molecular Chaperones: Lens α-Crystallins and Milk Caseins.

Molecular chaperone proteins perform a diversity of roles inside and outside the cell. One of the most important is the stabilization of misfolding proteins to prevent their aggregation, a process that is potentially detrimental to cell viability. Diseases such as Alzheimer's, Parkinson's, and cataract are characterized by the accumulation of protein aggregates. In vivo, many proteins are metastable and therefore under mild destabilizing conditions have an inherent tendency to misfold, aggregate, and hence lose functionality. As a result, protein levels are tightly regulated inside and outside the cell. Protein homeostasis, or proteostasis, describes the network of biological pathways that ensures the proteome remains folded and functional. Proteostasis is a major factor in maintaining cell, tissue, and organismal viability. We have extensively investigated the structure and function of intra- and extracellular molecular chaperones that operate in an ATP-independent manner to stabilize proteins and prevent their misfolding and subsequent aggregation into amorphous particles or highly ordered amyloid fibrils. These types of chaperones are therefore crucial in maintaining proteostasis under normal and stress (e.g., elevated temperature) conditions. Despite their lack of sequence similarity, they exhibit many common features, i.e., extensive structural disorder, dynamism, malleability, heterogeneity, oligomerization, and similar mechanisms of chaperone action. In this Account, we concentrate on the chaperone roles of α-crystallins and caseins, the predominant proteins in the eye lens and milk, respectively. Intracellularly, the principal ATP-independent chaperones are the small heat-shock proteins (sHsps). In vivo, sHsps are the first line of defense in preventing intracellular protein aggregation. The lens proteins αA- and αB-crystallin are sHsps. They play a crucial role in maintaining solubility of the crystallins (including themselves) with age and hence in lens proteostasis and, ultimately, lens transparency. As there is little metabolic activity and no protein turnover in the lens, crystallins are very long lived proteins. Lens proteostasis is therefore very different to that in normal, metabolically active cells. Crystallins undergo extensive post-translational modification (PTM), including deamidation, racemization, phosphorylation, and truncation, which can alter their stability. Despite this, the lens remains transparent for tens of years, implying that lens proteostasis is intimately integrated with crystallin PTMs. Many PTMs do not significantly alter crystallin stability, solubility, and functionality, which thereby facilitates lens transparency. In the long term, however, extensive accumulation of crystallin PTMs leads to large-scale crystallin aggregation, lens opacification, and cataract formation. Extracellularly, various ATP-independent molecular chaperones exist that exhibit sHsp-like structural and functional features. For example, caseins, the major milk proteins, exhibit chaperone ability by inhibiting the amorphous and amyloid fibrillar aggregation of a diversity of destabilized proteins. Caseins maintain proteostasis within milk by preventing deleterious casein amyloid fibril formation via incorporation of thousands of individual caseins into an amorphous structure known as the casein micelle. Hundreds of nanoclusters of calcium phosphate are sequestered within each casein micelle through interactions with short, highly phosphorylated casein sequences. This results in a stable biofluid that contains a high concentration of potentially amyloidogenic caseins and concentrations of calcium and phosphate that can be far in excess of the solubility of calcium phosphate. Casein micelle formation therefore performs vital roles in neonatal nutrition and calcium homeostasis in the mammary gland.

Sequence characteristics responsible for protein-protein interactions in the intrinsically disordered regions of caseins, amelogenins, and small heat-shock proteins.

Milk caseins and dental amelogenins are intrinsically disordered proteins (IDPs) that associate with themselves and others. Paradoxically, they are also described as hydrophobic proteins, which is difficult to reconcile with a solvent-exposed conformation. We attempt to resolve this paradox. We show that caseins and amelogenins are not hydrophobic proteins but they are more hydrophobic than most IDPs. Remarkably, uncharged residues from different regions of these mature proteins have a nearly constant average hydropathy but these regions exhibit different charged residue frequencies. A novel sequence analysis method was developed to identify hydrophobic and order-promoting regions that would favor conformational collapse. We found that such regions were uncommon; most hydrophobic and order-promoting residues were adjacent to hydrophilic or disorder-promoting residues. A further reason why caseins and amelogenins do not collapse is their high proportion of disorder-promoting proline residues. We conclude that in these proteins the hydrophobic effect is not large enough to cause conformational collapse but it can contribute, along with polar interactions, to protein-protein interactions. This behaviour is similar to the interaction of the disordered N-terminal region of small heat-shock proteins with either themselves during oligomer formation or other, unfolding, proteins during chaperone action.

Small Heat-shock Proteins Prevent α-Synuclein Aggregation via Transient Interactions and Their Efficacy Is Affected by the Rate of Aggregation.

The aggregation of α-synuclein (α-syn) into amyloid fibrils is associated with neurodegenerative diseases, collectively referred to as the α-synucleinopathies. In vivo, molecular chaperones, such as the small heat-shock proteins (sHsps), normally act to prevent protein aggregation; however, it remains to be determined how aggregation-prone α-syn evades sHsp chaperone action leading to its disease-associated deposition. This work examines the molecular mechanism by which two canonical sHsps, αB-crystallin (αB-c) and Hsp27, interact with aggregation-prone α-syn to prevent its aggregation in vitro Both sHsps are very effective inhibitors of α-syn aggregation, but no stable complex between the sHsps and α-syn was detected, indicating that the sHsps inhibit α-syn aggregation via transient interactions. Moreover, the ability of these sHsps to prevent α-syn aggregation was dependent on the kinetics of aggregation; the faster the rate of aggregation (shorter the lag phase), the less effective the sHsps were at inhibiting fibril formation of α-syn. Thus, these findings indicate that the rate at which α-syn aggregates in cells may be a significant factor in how it evades sHsp chaperone action in the α-synucleinopathies.

Deamidation of N76 in human γS-crystallin promotes dimer formation.

BACKGROUND: Cataract formation is often attributed to the build-up of post-translational modifications in the crystallin proteins of the eye lens. One such modification, the deamidation of N76 in human γS-crystallin to D76, is highly correlated with age-related cataract (Hooi et al. Invest. Ophthalmol. Vis. Sci. 53 (2012) 3554-3561). In the current work, this modification has been extensively characterised in vitro. METHODS: Biophysical characterisation was performed on wild type and N76D γS-crystallins using turbidity measurements to monitor aggregation, intrinsic fluorescence and circular dichroism spectroscopy to determine the folded state and NMR spectroscopy for identifying local changes in structure. Protein mass was determined using SEC-MALLS and analytical ultracentrifugation methods. RESULTS: Relative to the wild type protein, deamidation at N76 in γS-crystallin causes an increase in the thermal stability and resistance to thermally induced aggregation alongside a decrease in stability to denaturants, a propensity to aggregate rapidly once destabilised and a tendency to form a dimer. We ascribe the apparent increase in thermal stability upon deamidation to the formation of dimer which prevents the unfolding of the inherently less stable monomer. CONCLUSIONS: Deamidation causes a decrease in stability of γS-crystallin but this is offset by an increased tendency for dimer formation. GENERAL SIGNIFICANCE: Deamidation at N76 in human γS-crystallin likely has a combinatorial effect with other post-translational crystallin modifications to induce age-related cataract. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Monitoring Early-Stage Protein Aggregation by an Aggregation-Induced Emission Fluorogen.

Highly ordered protein aggregates, termed amyloid fibrils, are associated with a broad range of diseases, many of which are neurodegenerative, for example, Alzheimer's and Parkinson's. The transition from soluble, functional protein into insoluble amyloid fibril occurs via a complex process involving the initial generation of highly dynamic early stage aggregates or prefibrillar species. Amyloid probes, for example, thioflavin T and Congo red, have been used for decades as the gold standard for detecting amyloid fibrils in solution and tissue sections. However, these well-established dyes do not detect the presence of prefibrillar species formed during the early stages of protein aggregation. Prefibillar species have been proposed to play a key role in the cytotoxicity of amyloid fibrils and the pathogenesis of neurodegenerative diseases. Herein, we report a novel fluorescent dye (bis(triphenylphosphonium) tetraphenylethene (TPE-TPP)) with aggregation-induced emission characteristics for monitoring the aggregation process of amyloid fibrils. An increase in TPE-TPP fluorescence intensity is observed only with ordered protein aggregation, such as amyloid fibril formation, and not with stable molten globules states or amorphously aggregating species. Importantly, TPE-TPP can detect the presence of prefibrillar species formed early during fibril formation. TPE-TPP exhibits a distinctive spectral shift in the presence of prefibrillar species, indicating a unique structural feature of these intermediates. Using fluorescence polarization, which reflects the mobility of the emitting entity, the specific oligomeric pathways undertaken by various proteins during fibrillation could be discerned. Furthermore, we demonstrate the broad applicability of TPE-TPP to monitor amyloid fibril aggregation, including under diverse conditions such as at acidic pH and elevated temperature, or in the presence of amyloid inhibitors.

Coaggregation of κ-Casein and ß-Lactoglobulin Produces Morphologically Distinct Amyloid Fibrils.

The unfolding, misfolding, and aggregation of proteins lead to a variety of structural species. One form is the amyloid fibril, a highly aligned, stable, nanofibrillar structure composed of ß-sheets running perpendicular to the fibril axis. ß-Lactoglobulin (ß-Lg) and κ-casein (κ-CN) are two milk proteins that not only individually form amyloid fibrillar aggregates, but can also coaggregate under environmental stress conditions such as elevated temperature. The aggregation between ß-Lg and κ-CN is proposed to proceed via disulfide bond formation leading to amorphous aggregates, although the exact mechanism is not known. Herein, using a range of biophysical techniques, it is shown that ß-Lg and κ-CN coaggregate to form morphologically distinct co-amyloid fibrillar structures, a phenomenon previously limited to protein isoforms from different species or different peptide sequences from an individual protein. A new mechanism of aggregation is proposed whereby ß-Lg and κ-CN not only form disulfide-linked aggregates, but also amyloid fibrillar coaggregates. The coaggregation of two structurally unrelated proteins into cofibrils suggests that the mechanism can be a generic feature of protein aggregation as long as the prerequisites for sequence similarity are met.

A structural and functional study of Gln147 deamidation in αA-crystallin, a site of modification in human cataract.

Deamidation of Glu147 in human αA-crystallin is common in aged cataractous lenses (Hains and Truscott, Invest. Ophthalmol. Vis. Sci. 2010, 51, 3107). Accordingly, this modification may have a causative effect in cataract. αA-crystallin is a small heat-shock molecular chaperone protein that prevents aggregation of proteins and is the principal defence against crystallin unfolding and aggregation in the ageing lens. Deamidated Q147E αA-crystallin was structurally characterised using a variety of spectroscopic and biophysical methods, including NMR, circular dichroism and fluorescence spectroscopy and dynamic light scattering. The effect of Glu147 deamidation on αA-crystallin in vitro chaperone ability was determined for a variety of aggregating proteins. Compared to the wild type protein, Q147E αA-crystallin generally exhibited slightly reduced chaperone ability and a small loss of overall structure in its central α-crystallin domain while also showing significantly enhanced thermal stability and a tendency to form slightly larger oligomers. As αA-crystallin is the major lens protein, even a small loss of function could combine with other sources of age-related damage to the crystallins to contribute to lens opacification.

The structured core domain of αB-crystallin can prevent amyloid fibrillation and associated toxicity.

Mammalian small heat-shock proteins (sHSPs) are molecular chaperones that form polydisperse and dynamic complexes with target proteins, serving as a first line of defense in preventing their aggregation into either amorphous deposits or amyloid fibrils. Their apparently broad target specificity makes sHSPs attractive for investigating ways to tackle disorders of protein aggregation. The two most abundant sHSPs in human tissue are αB-crystallin (ABC) and HSP27; here we present high-resolution structures of their core domains (cABC, cHSP27), each in complex with a segment of their respective C-terminal regions. We find that both truncated proteins dimerize, and although this interface is labile in the case of cABC, in cHSP27 the dimer can be cross-linked by an intermonomer disulfide linkage. Using cHSP27 as a template, we have designed an equivalently locked cABC to enable us to investigate the functional role played by oligomerization, disordered N and C termini, subunit exchange, and variable dimer interfaces in ABC. We have assayed the ability of the different forms of ABC to prevent protein aggregation in vitro. Remarkably, we find that cABC has chaperone activity comparable to that of the full-length protein, even when monomer dissociation is restricted through disulfide linkage. Furthermore, cABC is a potent inhibitor of amyloid fibril formation and, by slowing the rate of its aggregation, effectively reduces the toxicity of amyloid-ß peptide to cells. Overall we present a small chaperone unit together with its atomic coordinates that potentially enables the rational design of more effective chaperones and amyloid inhibitors.

The Amyloid Fibril-Forming Properties of the Amphibian Antimicrobial Peptide Uperin†3.5.

The amphibian skin is a vast resource for bioactive peptides, which form the basis of the animals' innate immune system. Key components of the secretions of the cutaneous glands are antimicrobial peptides (AMPs), which exert their cytotoxic effects often as a result of membrane disruption. It is becoming increasingly evident that there is a link between the mechanism of action of AMPs and amyloidogenic peptides and proteins. In this work, we demonstrate that the broad-spectrum amphibian AMP uperin 3.5, which has a random-coil structure in solution but adopts an α-helical structure in membrane-like environments, forms amyloid fibrils rapidly in solution at neutral pH. These fibrils are cytotoxic to model neuronal cells in a similar fashion to those formed by the proteins implicated in neurodegenerative diseases. The addition of small quantities of 2,2,2-trifluoroethanol accelerates fibril formation by uperin 3.5, and is correlated with a structural stabilisation induced by this co-solvent. Uperin 3.5 fibril formation and the associated cellular toxicity are inhibited by the polyphenol (-)-epigallocatechin-3-gallate (EGCG). Furthermore, EGCG rapidly dissociates fully formed uperin 3.5 fibrils. Ion mobility-mass spectrometry reveals that uperin 3.5 adopts various oligomeric states in solution. Combined, these observations imply that the mechanism of membrane permeability by uperin 3.5 is related to its fibril-forming properties.

Real-time monitoring of amyloid growth in a rigid gel matrix.

We demonstrate the real-time monitoring of the growth of amyloid-protein aggregates in a semi-rigid gel environment constructed from a 5% w/v gelatin solution. The kinetics of amyloid fibril growth from reduced and carboxy-methylated κ-casein occurring in the gel medium was contrasted against that obtained in a regular solution assay. Aggregation kinetics were recorded using Thioflavin T fluorescence. Transmission electron microscopy was used to confirm the aggregates' existence and morphology. The current demonstration of controlled amyloid growth in a gel environment represents the first step towards development of an experimental model for investigating the role of spatial and medium factors in the kinetics of aggregation-based proteopathies.

Recognizing and analyzing variability in amyloid formation kinetics: Simulation and statistical methods.

We examine the phenomenon of variability in the kinetics of amyloid formation and detail methods for its simulation, identification and analysis. Simulated data, reflecting intrinsic variability, were produced using rate constants, randomly sampled from a pre-defined distribution, as parameters in an irreversible nucleation-growth kinetic model. Simulated kinetic traces were reduced in complexity through description in terms of three characteristic parameters. Practical methods for assessing convergence of the reduced parameter distributions were introduced and a bootstrap procedure was applied to determine convergence for different levels of intrinsic variation. Statistical methods for assessing the significance of shifts in parameter distributions, relating to either change in parameter mean or distribution shape, were tested. Robust methods for analyzing and interpreting kinetic data possessing significant intrinsic variance will allow greater scrutiny of the effects of anti-amyloid compounds in drug trials.

Protein aggregate turbidity: Simulation of turbidity profiles for mixed-aggregation reactions.

Due to their colloidal nature, all protein aggregates scatter light in the visible wavelength region when formed in aqueous solution. This phenomenon makes solution turbidity, a quantity proportional to the relative loss in forward intensity of scattered light, a convenient method for monitoring protein aggregation in biochemical assays. Although turbidity is often taken to be a linear descriptor of the progress of aggregation reactions, this assumption is usually made without performing the necessary checks to provide it with a firm underlying basis. In this article, we outline utilitarian methods for simulating the turbidity generated by homogeneous and mixed-protein aggregation reactions containing fibrous, amorphous, and crystalline structures. The approach is based on a combination of Rayleigh-Gans-Debye theory and approximate forms of the Mie scattering equations.