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The apolipoprotein family is a set of highly conserved proteins characterized by the presence of amphipathic α-helical sequences that mediate lipid binding. Paradoxically, this family of proteins is also prominent among the proteins known to form amyloid fibrils, characterized by extensive cross-β structure. Several apolipoproteins including apolipoprotein (apo) A-I, apoA-II and apoC-II accumulate in amyloid deposits of atherosclerotic lesions. This review illustrates the role of lipid-apolipoprotein interactions in apolipoprotein folding and aggregation with a specific focus on human apoC-II, a well-studied member of the family. In the presence of high concentrations of micellar lipid mimetics apoC-II adopts a stable and predominantly α-helical structure, similar to other members of the family and presumed to be the structure of apoC-II in circulating plasma lipoproteins. In contrast, lipid-free apoC-II aggregates to form long amyloid fibrils with a twisted ribbon-like morphology. Detailed structural analyses identify a letter G-like conformation as the basic building block within these fibrils. Phospholipids at submicellar concentrations accelerate apoC-II fibril formation by promoting the formation of a discrete tetrameric intermediate. Conversely, several small molecule lipid-mimetics inhibit apoC-II fibril formation at submicellar concentrations, inducing well-defined dimers unable to further aggregate. Finally, low concentrations of phospholipid micelles and bilayers induce the slow formation of amyloid fibrils with distinct rod-like fibril morphology. These studies highlight the diversity of lipid effects on apolipoprotein amyloid formation and reveal a conformational adaptability that could underlie the widespread occurrence of apolipoproteins in amyloid deposits and atheroma., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Human apolipoprotein (apo) C-II is one of several plasma apolipoproteins that form amyloid deposits in vivo and is an independent risk factor for cardiovascular disease. Lipid-free apoC-II readily self-assembles into twisted-ribbon amyloid fibrils but forms straight, rod-like amyloid fibrils in the presence of low concentrations of micellar phospholipids. Charge mutations exerted significantly different effects on rod-like fibril formation compared to their effects on twisted-ribbon fibril formation. For instance, the double mutant, K30D-D69K apoC-II, readily formed twisted-ribbon fibrils, while the rate of rod-like fibril formation in the presence of micellar phospholipid was negligible. Structural analysis of rod-like apoC-II fibrils, using hydrogen-deuterium exchange and NMR analysis showed exchange protection consistent with a core cross-β structure comprising the C-terminal 58-76 region. Molecular dynamics simulations of fibril arrangements for this region favoured a parallel cross-β structure. X-ray fibre diffraction data for aligned rod-like fibrils showed a major meridional spacing at 4.6 Å and equatorial spacings at 9.7, 23.8 and 46.6 Å. The latter two equatorial spacings are not observed for aligned twisted-ribbon fibrils and are predicted for a model involving two cross-β fibrils in an off-set antiparallel structure with four apoC-II units per rise of the β-sheet. This model is consistent with the mutational effects on rod-like apoC-II fibril formation. The lipid-dependent polymorphisms exhibited by apoC-II fibrils could determine the properties of apoC-II in renal amyloid deposits and their potential role in the development of cardiovascular disease., (© 2018 Federation of European Biochemical Societies.)

The apolipoprotein family is structurally defined by amphipathic α-helical regions that interact with lipid surfaces. In the absence of lipid, human apolipoprotein (apo) C-II also forms well-defined amyloid fibrils with cross-β structure. Formation of this β-structure is accompanied by the burial of two charged residues, K30 and D69, that form an ion-pair within the amyloid fibril core. Molecular dynamics (MD) simulations indicate these buried residues form both intra- and intersubunit ion-pair interactions that stabilize the fibril. Mutations of the ion-pair (either K30D or D69K) reduce fibril stability and prevent fibril formation by K30D apoC-II under standard conditions. We investigated whether mixing K30D apoC-II with other mutants would overcome this loss of fibril forming ability. Co-incubation of equimolar mixtures of K30D apoC-II with wild-type, D69K, or double-mutant (K30D/D69K) apoC-II promoted the incorporation of K30D apoC-II into hybrid fibrils with increased stability. MD simulations showed an increase in the number of intersubunit ion-pair interactions accompanied the increased stability of the hybrid fibrils. These results demonstrate the important role of both intra- and intersubunit charge interactions in stabilizing apoC-II amyloid fibrils, a process that may be a key factor in determining the general ability of proteins to form amyloid fibrils.

Apolipoproteins form amphipathic helical structures that bind lipid surfaces. Paradoxically, lipid-free apolipoproteins display a strong propensity to form cross-β structure and self-associate into disease-related amyloid fibrils. Studies of apolipoprotein C-II (apoC-II) amyloid fibrils suggest that a K30-D69 ion pair accounts for the dual abilities to form helix and cross-β structure. Consistent with this is the observation that a K30D mutation prevents fibril formation under standard fibril forming conditions. However, we found that fibril formation by K30D apoC-II proceeded readily at low pH and a higher salt or protein concentration. Structural analysis demonstrated that K30D apoC-II fibrils at pH 7 have a structure similar to that of the wild-type fibrils but are less stable. Molecular dynamics simulations of the wild-type apoC-II fibril model at pH 7 and 3 showed that the loss of charge on D69 at pH 3 leads to greater separation between residues K30 and D69 within the fibril with a corresponding reduction in β-strand content around residue 30. In contrast, in simulations of the K30D mutant model at pH 7 and 3, residues D30 and D69 moved closer at pH 3, accompanied by an increase in β-strand content around residue 30. The simulations also demonstrated a strong dominance of inter- over intramolecular contacts between ionic residues of apoC-II and suggested a cooperative mechanism for forming favorable interactions between the individual strands under different conditions. These observations demonstrate the important role of the buried K30-D69 ion pair in the stability and solution properties of apoC-II amyloid fibrils.

Plasma apolipoproteins form amphipathic α helices in lipid environments but in the lipid-free state show a high propensity to form β structure and self-associate into amyloid fibrils. The widespread occurrence of apolipoproteins in amyloid plaques suggests disease-related roles, specifically in atherosclerosis. To reconcile the dual abilities of apolipoproteins to form either α helices or cross-β sheet structures, we examined fibrils formed by human apolipoprotein C-II (apoC-II). A structural model for apoC-II fibrils shows a cross-β core with parallel β strands, including a buried K30-D69 charge pair. We investigated the effect of abolishing this charge pair in mutant D69K apoC-II. Fluorescence studies indicated more rapid fibril formation and less solvent accessibility of tryptophan (W26) in D69K apoC-II fibrils than in wild-type (WT) fibrils. X-ray diffraction data of aligned D69K apoC-II fibrils yielded a typical cross-β structure with increased β sheet spacing compared to that of WT fibrils. Hydrogen/deuterium (H/D) exchange patterns were similar for D69K apoC-II fibrils compared to WT fibrils, albeit with an overall reduction in the level of slow H/D exchange, particularly around residues 29-32. Molecular dynamics simulations indicated reduced β strand content for a model D69K apoC-II tetramer compared to the WT tetramer and confirmed an expansion of the cross-β spacing that contributed to the formation of a stable charge pair between K69 and E27. The results highlight the importance of charge-pair interactions within the apoC-II fibril core, which together with numerous salt bridges in the flexible connecting loop play a major role in the ability of lipid-free apoC-II to form stable cross-β fibrils.

Apolipoprotein A-I (apoA-I) is the major component of high density lipoproteins and plays a vital role in reverse cholesterol transport. Lipid-free apoA-I is the main constituent of amyloid deposits found in atherosclerotic plaques, an acquired type of amyloidosis, whereas its N-terminal fragments have been associated with a hereditary form, known as familial apoA-I amyloidosis. Here, we identified and verified four "aggregation-prone" segments of apoA-I with amyloidogenic properties, utilizing electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy and polarized light microscopy. These segments may act as conformational switches, possibly controlling the transition of the α-helical apoA-I content into the "cross-β" architecture of amyloid fibrils. A structural model illuminating the structure of amyloid fibrils formed by the N-terminal fragments of apoA-I is proposed, indicating that two of the identified chameleon segments may play a vital part in the formation of amyloid fibrils in familial apoA-I amyloidosis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Protein misfolding and aggregation, leading to amyloid fibril formation, are characteristic of many devastating and debilitating amyloid diseases. Accordingly, there is significant interest in the mechanisms underlying amyloid fibril formation and identification of possible intervention tools. Small molecule drug compounds approved for human use or for use in phase I-III clinical trials were investigated for their effects on amyloid formation by human apolipoprotein (apo) C-II. Several of these compounds modulated the rate of amyloid formation by apoC-II. Epigallocatechin gallate (EGCG), a green tea catechin, was an effective inhibitor of apoC-II fibril formation, and the antipsychotic drug, fluphenazine·HCl, was a potent activator. Both EGCG and fluphenazine·HCl exerted concentration-dependent effects on the rate of fibril formation, bound to apoC-II fibrils with high affinity, and competitively reduced thioflavin T binding. EGCG significantly altered the size distribution of fibrils, most likely by promoting the lateral association of fibrils and subsequent formation of large aggregates. Fluphenazine·HCl did not significantly alter the size distribution of fibrils, but it may induce the formation of a small population of rod-like fibrils that differ from the characteristic ribbon-like fibrils normally observed for apoC-II. The findings of this study emphasize the effects of small molecule drugs on the kinetics of amyloid fibril formation and their roles in determining fibril structure and aggregate size.

The misfolding, aggregation, and accumulation of proteins as amyloid fibrils is a defining characteristic of several debilitating diseases. Human apolipoprotein C-II (apoC-II) amyloid fibrils are representative of the fibrils formed by a number of plasma apolipoproteins implicated in amyloid-related disease. Previous structural analyses identified a buried charge pair between residues K30 and D69 within apoC-II amyloid fibrils. We have investigated the effects of amino acid substitutions of these residues on apoC-II fibril formation. Two point mutations of apoC-II, D69K and K30D, as well as a reversed ion-pair mutant containing both mutations (KDDK) were generated. Fibril formation by the double mutant, apoC-II KDDK, and apoC-II D69K was enhanced compared to that of wild-type (WT) apoC-II, while apoC-II K30D lacked the ability to form fibrils under standard conditions. Structural analyses showed that WT apoC-II, apoC-II D69K, and apoC-II KDDK fibrils have similar secondary structures and morphologies. Size distribution analyses revealed that apoC-II D69K fibrils have a broader range of fibril sizes while apoC-II KDDK fibrils showed an increased frequency of closed fibrillar loops. ApoC-II D69K fibrils exhibited reduced thioflavin T binding capacity compared to that of fibrils formed by WT apoC-II and apoC-II KDDK. These results indicate that specific charge and charge-pair mutations within apoC-II significantly alter the ability to form fibrils and that position 69 within apoC-II plays a key role in the rate-limiting step of apoC-II fibril formation.

Amyloid fibrils result from the self-assembly of proteins into large aggregates with fibrillar morphology and common structural features. These fibrils form the major component of amyloid plaques that are associated with a number of common and debilitating diseases, including Alzheimer's disease. While a range of unrelated proteins and peptides are known to form amyloid fibrils, a common feature is the formation of aggregates of various sizes, including mature fibrils of differing length and/or structural morphology, small oligomeric precursors, and other less well-understood forms such as amorphous aggregates. These various species can possess distinct biochemical, biophysical, and pathological properties. Sedimentation velocity analysis can characterize amyloid fibril formation in exceptional detail, providing a particularly useful method for resolving the complex heterogeneity present in amyloid systems. In this chapter, we describe analytical methods for accurate quantification of both total amyloid fibril formation and the formation of distinct amyloid structures based on differential sedimentation properties. We also detail modern analytical ultracentrifugation methods to determine the size distribution of amyloid aggregates. We illustrate examples of the use of these techniques to provide biophysical and structural information on amyloid systems that would otherwise be difficult to obtain., (© 2015 Elsevier Inc. All rights reserved.)

Apolipoproteins are a key component of lipid transport in the circulatory system and share a number of structural features that facilitate this role. When bound to lipoprotein particles, these proteins are relatively stable. However, in the absence of lipids they display conformational instability and a propensity to aggregate into amyloid fibrils. Apolipoprotein C-II (apoC-II) is a member of the apolipoprotein family that has been well characterised in terms of its misfolding and aggregation. In the absence of lipid, and at physiological ionic strength and pH, apoC-II readily forms amyloid fibrils with a twisted ribbon-like morphology that are amenable to a range of biophysical and structural analyses. Consistent with its lipid binding function, the misfolding and aggregation of apoC-II are substantially affected by the presence of lipid. Short-chain phospholipids at submicellar concentrations significantly accelerate amyloid formation by inducing a tetrameric form of apoC-II that can nucleate fibril aggregation. Conversely, phospholipid micelles and bilayers inhibit the formation of apoC-II ribbon-type fibrils, but induce slow formation of amyloid with a distinct straight fibril morphology. Our studies of the effects of lipid at each stage of amyloid formation, detailed in this chapter, have revealed complex behaviour dependent on the chemical nature of the lipid molecule, its association state, and the protein:lipid ratio.

The tumour-derived monoclonal IgM antibody PAT-SM6 specifically kills malignant cells by an apoptotic mechanism linked to the excessive uptake of plasma lipids. The mechanism is postulated to occur via the multi-point attachment of PAT-SM6 to the unfolded protein response regulator GRP78, located on the surface of tumour cells, coupled to the simultaneous binding of plasma low density lipoprotein (LDL). We prepared and characterised LDL and oxidized LDL using sedimentation velocity and small-angle X-ray scattering (SAXS) analysis. Enzyme-linked immunosorbent (ELISA) techniques indicated apparent dissociation constants of approximately 20 nM for the binding of LDL or oxidized LDL to PAT-SM6. ELISA experiments showed cross competition with LDL inhibiting PAT-SM6 binding to immobilised GRP78, while, in the reverse experiment, GRP78 inhibited PAT-SM6 binding to immobilized LDL. In contrast to the results of the ELISA experiments, sedimentation velocity experiments indicated relatively weak interactions between LDL and PAT-SM6, suggesting immunoabsorbance to the microtiter plate is driven by an avidity-based binding mechanism. The importance of avidity and the multipoint attachment of antigens to PAT-SM6 was further investigated using antigen-coated polystyrene beads. Absorption of GRP78 or LDL to polystyrene microspheres led to an increase in the inhibition of PAT-SM6 binding to microtiter plates coated with GRP78 or LDL, respectively. These results support the hypothesis that the biological action of PAT-SM6 in tumour cell apoptosis depends on the multivalent nature of PAT-SM6 and the ability to interact simultaneously with LDL and multiple GRP78 molecules clustered on the tumour cell surface.

The in vivo aggregation of proteins into amyloid fibrils suggests that cellular mechanisms that normally prevent or reverse this aggregation have failed. The small heat-shock molecular chaperone protein αB-crystallin (αB-c) inhibits amyloid formation and colocalizes with amyloid plaques; however, the physiological reason for this localization remains unexplored. Here, using apolipoprotein C-II (apoC-II) as a model fibril-forming system, we show that αB-c binds directly to mature amyloid fibrils (Kd 5.4 ± 0.5 μM). In doing so, αB-c stabilized the fibrils from dilution-induced fragmentation, halted elongation of partially formed fibrils, and promoted the dissociation of mature fibrils into soluble monomers. Moreover, in the absence of dilution, the association of αB-c with apoC-II fibrils induced a 14-fold increase in average aggregate size, resulting in large fibrillar tangles reminiscent of protein inclusions. We propose that the binding of αB-c to fibrils prevents fragmentation and mediates the lateral association of fibrils into large inclusions. We further postulate that transient interactions of apoC-II with αB-c induce a fibril-incompetent monomeric apoC-II form, preventing oligomerization and promoting fibril dissociation. This work reveals previously unrecognized mechanisms of αB-c chaperone action in amyloid assembly and fibril dynamics, and provides a rationale for the in vivo colocalization of small heat-shock proteins with amyloid deposits.-Binger, K. J., Ecroyd, H., Yang, S., Carver, J. A., Howlett, G. J., Griffin, M. D. W. Avoiding the oligomeric state: αB-crystallin inhibits fragmentation and induces dissociation of apolipoprotein C-II amyloid fibrils.

Expression of the periplasmic protein PcoE of Escherichia coli is induced strongly by cupric salts under the control of the chromosomal copper tolerance system cusRS. Its isolation and study were complicated by de-amidation of Asn 54 and 103 at alkaline pH. Its apo form is essentially unstructured in solution and can be likened to a large unstructured multidentate ligand carrying multiple metal binding sites (15 Met; 10 His; 13 Asp, Glu; 10 Asn; 6 Lys). As expected, it binds multiple soft metal ions Cu(+) and Ag(+) non-cooperatively with the highest affinity for Cu(I) in the picomolar range (K(D)~10(-12) M). Binding of multiple soft ions induced dimerization and formation of some α-helical structure. PcoE also binds the harder metal ions Cu(2+) or Zn(2+) but with lower affinities and in smaller numbers. Cu(II) bound in PcoE is reduced readily to more tightly bound Cu(I). Overall, these properties mean that it is difficult to characterize individual species of defined metal content. Similar properties and difficulties have been reported for the homologous silver-binding protein SilE from Salmonella. However, the properties are consistent with a role for PcoE as a 'metal sponge' acting as a first line of defence against metal toxicity (under the control of the copper tolerance system cusRS) until the copper resistance operon pcoABCD is expressed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Amyloid fibrils and their soluble oligomeric intermediates are implicated in several age-related diseases including Alzheimer's and Parkinson's diseases. The distribution of oligomers and fibrils is related to toxicity and is dependent on the pathways for fibril assembly, generally considered to occur via a slow nucleation step that precedes fibril elongation. Human apolipoprotein (apo) C-II forms amyloid fibrils via a reversible self-assembly process accompanied by closed-loop formation and fibril breaking and joining. Our fluorescence quenching and sedimentation velocity experiments with Alexa488-labeled apoC-II indicated a time-dependent subunit interchange for both linear and closed-loop fibrils, while dilution experiments using mature fibrils indicated a shift to smaller size distributions consistent with a reversible assembly pathway. To account for this behavior, we developed an equilibrium self-association model that describes the final size distributions of apoC-II fibrils formed at different starting concentrations. The model proposes a reversible isomerization of apoC-II monomer to form an active conformer that self-assembles into fibrils via an isodesmic self-association pathway coupled to fibril length-dependent closed-loop formation. The model adequately described fibril size distributions and the proportion of closed loops as a function of total apoC-II concentration over the concentration range 0.1-0.5 mg/ml. Extension of the model to include the rates of isomerization, self-association and fibril breaking and joining provided satisfactory global fits to kinetic data on fibril formation and changes in average fibril size at different apoC-II starting concentrations. The model provides a simple thermodynamic description of the processes governing the size distribution of apoC-II fibrils at equilibrium and the formation of discrete oligomeric intermediates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Apolipoprotein A-I (apoA-I) is deposited as amyloid within various major organs in hereditary apoA-I amyloidosis, and in arterial plaques associated with atherosclerosis. We have identified a tryptic fragment of apoA-I, apoA-I(46-59), that retains the ability to form amyloid-like fibrils with cross-β structure. ApoA-I(46-59) corresponds closely to a conformationally extended segment in the crystal structure of apoA-IΔ(185-243) and is located in the N-terminal region of apoA-I, which accumulates in hereditary apoA-I amyloidosis. Our results provide direct experimental evidence that this region of apoA-I is amyloidogenic and integral to initiation and propagation of amyloid formation by the protein., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

The misfolding and aggregation of proteins to form amyloid fibrils is a characteristic feature of several common age-related diseases. Agents that directly inhibit formation of amyloid fibrils represent one approach to combating these diseases. We have investigated the potential of a cyclic peptide to inhibit fibril formation by fibrillogenic peptides from human apolipoprotein C-II (apoC-II). Cyc[60-70] was formed by disulfide cross-linking of cysteine residues added to the termini of the fibrillogenic peptide comprising apoC-II residues 60-70. This cyclic peptide did not self-associate into fibrils. However, substoichiometric concentrations of cyc[60-70] significantly delayed fibril formation by the fibrillogenic, linear peptides apoC-II[60-70] and apoC-II[56-76]. Reduction of the disulfide bond or scrambling the amino acid sequence within cyc[60-70] significantly impaired its inhibitory activity. The solution structure of cyc[60-70] was solved using NMR spectroscopy, revealing a well-defined structure comprising a hydrophilic face and a more hydrophobic face containing the Met60, Tyr63, Ile66 and Phe67 side chains. Molecular dynamics (MD) studies identified a flexible central region within cyc[60-70], while MD simulations of "scrambled" cyc[60-70] indicated an increased formation of intramolecular hydrogen bonds and a reduction in the overall flexibility of the peptide. Our structural studies suggest that the inhibitory activity of cyc[60-70] is mediated by an elongated structure with inherent flexibility and distinct hydrophobic and hydrophilic faces, enabling cyc[60-70] to interact transiently with fibrillogenic peptides and inhibit fibril assembly. These results suggest that cyclic peptides based on amyloidogenic core peptides could be useful as specific inhibitors of amyloid fibril formation., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The monoclonal IgM antibody PAT-SM6 derived from human tumours induces apoptosis in tumour cells and is considered a potential anti-cancer agent. A primary target for PAT-SM6 is the unfolded protein response regulator GRP78, over-expressed externally on the cell surface of tumour cells. Small angle X-ray scattering (SAXS) studies of human GRP78 showed a two-domain dumbbell-shaped monomer, while SAXS analysis of PAT-SM6 revealed a saucer-shaped structure accommodating five-fold symmetry, consistent with previous studies of related proteins. Sedimentation velocity analysis of GRP78 and PAT-SM6 mixtures indicated weak complex formation characterized by dissociation constants in the high micromolar concentration range. In contrast, enzyme-linked immunosorbant assays (ELISAs) showed strong and specific interactions between PAT-SM6 and immobilized GRP78. The apparent binding constant estimated from a PAT-SM6 saturation curve correlated strongly with the concentration of GRP78 used to coat the microtiter tray. Experiments using polyclonal antiGRP78 IgG antibodies or a monoclonal IgG derivative of PAT-SM6 did not show a similar dependence. Competition experiments with soluble GRP78 indicated more effective inhibition of PAT-SM6 binding at low GRP78 coating concentrations. These observations suggest an avidity-based binding mechanism that depends on the multi-point attachment of PAT-SM6 to GRP78 clustered on the surface of the tray. Analysis of ELISA data at high GRP78 coating concentrations yielded an apparent dissociation constant of approximately 4 nM. We propose that the biological action of PAT-SM6 in tumour cell apoptosis may depend on the multivalent nature of PAT-SM6 and the high avidity of its interaction with multiple GRP78 molecules clustered on the tumour cell surface.

Human apolipoprotein (apo) C-II is one of several lipid-binding proteins that self-assemble into fibrils and accumulate in disease-related amyloid deposits. A general characteristic of these amyloid deposits is the presence of lipids, known to modulate individual steps in amyloid fibril formation. ApoC-II fibril formation is activated by submicellar phospholipids but inhibited by micellar lipids. We examined the mechanism for the activation by submicellar lipids using the fluorescently labeled, short-chain phospholipid 1-dodecyl-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-2-hydroxyglycero-3-phosphocholine (NBD-lyso-12-PC). Addition of submicellar NBD-lyso-12-PC increased the rate of fibril formation by apoC-II approximately 2-fold. Stopped flow kinetic analysis using fluorescence detection and low, non-fibril-forming concentrations of apoC-II indicated NBD-lyso-12-PC binds rapidly, on the millisecond time scale, followed by the slower formation of discrete apoC-II tetramers. Sedimentation velocity analysis showed NBD-lyso-12-PC binds to both apoC-II monomers and tetramers at approximately five sites per monomer with an average dissociation constant of approximately 10 μM. Mature apoC-II fibrils formed in the presence of NBD-lyso-12-PC were devoid of lipid, indicating a purely catalytic role for submicellar lipids in the activation of apoC-II fibril formation. These studies demonstrate the catalytic potential of small amphiphilic molecules in controlling protein folding and fibril assembly pathways.

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold. Fibrils produced at shear rates of 150 and 300 s(-1) were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s(-1), tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 μg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.

The assembly of proteins into large fibrillar aggregates, known as amyloid fibrils, is associated with a number of common and debilitating diseases. In some cases, proteins deposit extracellularly, while in others the aggregation is intracellular. A common feature of these diseases is the presence of aggregates of different sizes, including mature fibrils, small oligomeric precursors, and other less well understood structural forms such as amorphous aggregates. These various species possess distinct biochemical, biophysical, and pathological properties. Here, we detail a number of techniques that can be employed to examine amyloid fibrils and oligomers using a fluorescence-detection system (FDS) coupled with the analytical ultracentrifuge. Sedimentation velocity analysis using fluorescence detection is a particularly useful method for resolving the complex heterogeneity present in amyloid systems and can be used to characterize aggregation in exceptional detail. Furthermore, the fluorescence detection module provides a number of particularly attractive features for the analysis of aggregating proteins. It expands the practical range of concentrations of aggregating proteins under study, which is useful for greater insight into the aggregation process. It also enables the assessment of aggregation behavior in complex biological solutions, such as cell lysates, and the assessment of processes that regulate in-cell or extracellular aggregation kinetics. Four methods of fluorescent detection that are compatible with the current generation of FDS instrumentation are described: (1) Detection of soluble amyloid fibrils using a covalently bound fluorophore. (2) Detection of amyloid fibrils using an extrinsic dye that emits fluorescence when bound to fibrils. (3) Detection of fluorescently-labeled lipids and their interaction with oligomeric amyloid intermediates. (4) Detection of green fluorescence protein (GFP) constructs and their interactions within mammalian cell lysates., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The misfolding and aggregation of proteins to form amyloid fibrils are associated with a number of debilitating, age-related diseases. Many of the proteins that form amyloid in vivo are lipid-binding proteins, accounting for the significant impact of lipids on the rate of formation and morphology of amyloid fibrils. To systematically investigate the effect of lipid-like compounds, we screened a range of amphipathic lipids and detergents for their effect on amyloid fibril formation by human apolipoprotein (apo) C-II. The initial screen, conducted using a set of amphiphiles at half critical micelle concentration, identified several activators and inhibitors that were selected for further analysis. Sedimentation analysis and circular dichroism studies of apoC-II at low, non-fibril-forming concentrations (0.05 mg/ml) revealed that all of the inhibitors induced the formation of apoC-II dimers enriched in α-helical content while the activators promoted the formation of stable apoC-II tetramers with increased β-structure. Kinetic analysis identified modulators of apoC-II fibril formation that were effective at concentrations as low as 10 μM, corresponding to a modulator-to-apoC-II ratio of approximately 1:10. Delayed addition of the test compounds after fibril formation had commenced allowed the effects of selected amphiphiles on fibril elongation to be determined separately from their effects on fibril nucleation. The results indicated that specific amphiphiles induce structural changes in apoC-II that cause separate and independent effects on fibril nucleation and elongation. Low-molecular-weight amphipathic lipids and detergents may serve as useful, stage-specific modulators of protein self-assembly and fibril formation in disease-prevention strategies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The self-assembly of specific proteins to form insoluble amyloid fibrils is a characteristic feature of a number of age-related and debilitating diseases. Lipid-free human apolipoprotein C-II (apoC-II) forms characteristic amyloid fibrils and is one of several apolipoproteins that accumulate in amyloid deposits located within atherosclerotic plaques. X-ray diffraction analysis of aligned apoC-II fibrils indicated a simple cross-β-structure composed of two parallel β-sheets. Examination of apoC-II fibrils using transmission electron microscopy, scanning transmission electron microscopy, and atomic force microscopy indicated that the fibrils are flat ribbons composed of one apoC-II molecule per 4.7-Å rise of the cross-β-structure. Cross-linking results using single-cysteine substitution mutants are consistent with a parallel in-register structural model for apoC-II fibrils. Fluorescence resonance energy transfer analysis of apoC-II fibrils labeled with specific fluorophores provided distance constraints for selected donor-acceptor pairs located within the fibrils. These findings were used to develop a simple 'letter-G-like' β-strand-loop-β-strand model for apoC-II fibrils. Fully solvated all-atom molecular dynamics (MD) simulations showed that the model contained a stable cross-β-core with a flexible connecting loop devoid of persistent secondary structure. The time course of the MD simulations revealed that charge clusters in the fibril rearrange to minimize the effects of same-charge interactions inherent in parallel in-register models. Our structural model for apoC-II fibrils suggests that apoC-II monomers fold and self-assemble to form a stable cross-β-scaffold containing relatively unstructured connecting loops., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures. The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases. A number of plasma apolipoproteins, including apolipoprotein (apo) A-I, apoA-II, apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins. We review present knowledge of amyloid formation by apolipoproteins in disease, with particular focus on atherosclerosis. Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions. Additionally, we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis, and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.

The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.

Analytical ultracentrifugation is a classical technique used to study the solution behavior of proteins. Experimentally determined sedimentation coefficients provide information regarding the size, shape, and interactions of biological macromolecules. Sedimentation velocity methods have been used to characterize the different aggregation states of amyloid oligomers and fibrils. This chapter first describes the theoretical background for sedimentation velocity analysis. It then provides experimental protocols for sedimentation velocity experiments using the analytical ultracentrifuge. Finally, this chapter describes the procedure used to analyze sedimentation velocity data to obtain the size distribution of amyloid fibrils and their oligomeric precursors.

Using experimental and computational methods we identified the effects of mutation on the structure and dynamics of the amyloidogenic peptide apoC-II(60-70), in monomeric and oligomeric states. Methionine (Met60) substitutions to hydrophilic Gln, hydrophobic Val, and methionine sulfoxide residues were investigated and the results compared with observations of fibril formation by the wild-type, Met60Gln, Met60Val, and oxidised Met60 (oxi-Met) apoC-II(60-70) peptides. ThT fluorescence measurements showed fibril formation by all peptides, however with different kinetics. The wild-type and Met60Val peptides formed fibrils fastest, while oxi-Met and Met60Gln peptides exhibited significantly longer lag phases. Molecular dynamics simulations showed that the mutated monomers exhibited structural features consistent with fibril-forming propensity, such as β-hairpin conformation and a hydrophobic core. However, important differences to the wild-type were also noted, such as increased structural flexibility (oxi-Met and Met60Gln systems) and a broader distribution of the aromatic angle orientation, which could contribute to the different fibrillation kinetics observed in these peptides. Our results also showed that the critical nucleus size for fibril formation by apoC-II(60-70) may not be very large, since tetrameric oligomers in anti-parallel configuration were very stable within the 100 ns of simulations. The single-point mutations Met60Val and Met60Gln had no significant effect on the structural stability of the tetramer. The rate of fibril formation by apoC-II(60-70) peptides was generally much faster compared to longer apoC-II(56-76) peptides. Also, the effects of amino acid modifications on the kinetics of peptide fibril formation differ from the effects observed for apoC-II(56-76) and full-length apoC-II, suggesting that additional mechanisms are involved in fibril formation by mature apoC-II.

Amyloid fibrils and their oligomeric intermediates accumulate in several age-related diseases where their presence is considered to play an active role in disease progression. A common characteristic of amyloid fibril formation is an initial lag phase indicative of a nucleation-elongation mechanism for fibril assembly. We have investigated fibril formation by human apolipoprotein (apo) C-II. ApoC-II readily forms amyloid fibrils in a lipid-dependent manner via an initial nucleation step followed by fibril elongation, breaking, and joining. We used fluorescence techniques and stopped-flow analysis to identify the individual kinetic steps involved in the activation of apoC-II fibril formation by the short-chain phospholipid dihexanoyl phosphatidylcholine (DHPC). Submicellar DHPC activates fibril formation by promoting the rapid formation of a tetrameric species followed by a slow isomerisation that precedes monomer addition and fibril growth. Global fitting of the concentration dependence of apoC-II fibril formation showed that DHPC increased the overall tetramerisation constant from 7.5 x 10(-13) to 1.2 x 10(-6) microM(-3) without significantly affecting the rate of fibril elongation, breaking, or joining. Studies on the effect of DHPC on the free pool of apoC-II monomer and on fibril formation by cross-linked apoC-II dimers further demonstrate that DHPC affects nucleation but not elongation. These studies demonstrate the capacity of small lipid compounds to selectively target individual steps in the amyloid fibril forming pathway., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A common feature of many amyloid diseases is the appearance of oxidized, aggregated proteins. Methionine is one of the most readily oxidized amino acids, and its oxidative state is regulated in vivo by the methionine sulfoxide reductases (Msr). Here, we have explored the basis by which methionine oxidation is linked to amyloid disease by comparing the reduction of oxidized amyloid fibrils and monomer. We show that oxidized amyloid fibrils are not as effectively reduced by the Msr enzymes as the monomer. This work suggests a mechanism by which oxidized proteins and aggregates can accumulate as a part of degenerative disease.

Apolipoprotein A-I (apoA-I) is the major protein component of HDL, where it plays an important role in cholesterol transport. The deposition of apoA-I derived amyloid is associated with various hereditary systemic amyloidoses and atherosclerosis; however, very little is known about the mechanism of apoA-I amyloid formation. Methionine residues in apoA-I are oxidized via several mechanisms in vivo to form methionine sulfoxide (MetO), and significant levels of methionine oxidized apoA-I (MetO-apoA-I) are present in normal human serum. We investigated the effect of methionine oxidation on the structure, stability, and aggregation of full-length, lipid-free apoA-I. Circular dichrosim spectroscopy showed that oxidation of all three methionine residues in apoA-I caused partial unfolding of the protein and decreased its thermal stability, reducing the melting temperature (T(m)) from 58.7 degrees C for native apoA-I to 48.2 degrees C for MetO-apoA-I. Analytical ultracentrifugation revealed that methionine oxidation inhibited the native self association of apoA-I to form dimers and tetramers. Incubation of MetO-apoA-I for extended periods resulted in aggregation of the protein, and these aggregates bound Thioflavin T and Congo Red. Inspection of the aggregates by electron microscopy revealed fibrillar structures with a ribbon-like morphology, widths of approximately 11 nm, and lengths of up to several microns. X-ray fibre diffraction studies of the fibrils revealed a diffraction pattern with orthogonal peaks at spacings of 4.64 A and 9.92 A, indicating a cross-beta amyloid structure. This systematic study of fibril formation by full-length apoA-I represents the first demonstration that methionine oxidation can induce amyloid fibril formation.

Objectives: Amyloid fibrils and amyloid-like structures are implicated in atherosclerosis via macrophage activation and inflammation. A common property of amyloid-like structures is their ability to induce thioflavin T (ThT) fluorescence. We measured ThT fluorescence in serum and related these levels to traditional cardiovascular risk factors and non-invasive measures of vascular dysfunction (elasticity). In addition, chemically modified serum components that contribute to serum ThT fluorescence were explored and identified., Design, Methods, and Results: Sera from 105 people, including 35 healthy subjects, and 70 high cardiovascular risk patients (36 with rheumatoid arthritis and 34 with systemic lupus erythrematosus) showed an 8.75-fold variation in induced ThT fluorescence. Although mean (+/-SD) ThT fluorescence did not differ significantly between groups (controls 0.97+/-0.26, RA 1.12+/-0.45, and SLE 0.74+/-0.23), the combined data set showed significant inverse correlation (p=0.046) between ThT fluorescence tertiles and small artery elasticity. Correlation was also found between ThT fluorescence tertiles and LDL-cholesterol, total-cholesterol, and C-reactive protein. Floatation fractionation of apoB containing lipoproteins showed that ThT reactivity in this fraction correlated with both serum oxidised-LDL and LDL-cholesterol levels. However, approximately 94% of ThT reactivity in serum was associated with the non-apoB containing serum fraction, with the majority of ThT fluorescence associated with albumin. Incubation of purified albumin with glucose or with methylglyoxal induced ThT fluorescence, suggesting that glycated or chemical adducts of albumin contribute to the variation in ThT fluorescence of human serum., Conclusions: We propose that the detection of these adducts in serum using ThT fluorescence measurements may provide a marker for chemically modified protein structures that could assist the assessment of cardiovascular disease risk., ((c) 2009 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

The oxidation of methionine residues in proteins can inhibit the self-assembly of proteins to form amyloid fibrils. For human apolipoprotein (apo) C-II the oxidation of methionine at position 60 inhibits fibril formation by the mature protein and by the core peptides apoC-II(56-76) and apoC-II(60-70). To investigate the molecular nature of these effects, we carried out fully solvated, all-atom molecular dynamics simulations of the structural changes in apoC-II(56-76) associated with substitutions of oxidized methionine (Met ox) at position 60. The results with apoC-II(56-76) (Met ox) showed less flexibility in structure, leading to a perturbation of the hydrophobic core. Valine substitution at position 60 showed an increased tendency to explore a wide range of conformational space, whereas the behavior of the Gln substitution mutant was similar to the wild-type peptide. These simulations are consistent with kinetic measurements which showed that a Met60Gln substitution within apoC-II(56-76) had little effect on the rate of fibril formation whereas substitution of Met ox or Val at position 60 lead to significant inhibition of peptide fibril formation. The effects of amino acid modification and substitutions on the kinetics of peptide fibril formation differ from the effects observed with full-length apoC-II inferring that additional mechanisms are involved in fibril formation by mature apoC-II.

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies has confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying beta-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular beta-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

We investigated the effect of submicellar lipids on amyloid fibril formation. Thioflavin T fluorescence studies showed that submicellar levels of the short-chain phospholipids, dipentanoylphosphatidylcholine and dihexanoylphosphatidylcholine, strongly inhibited amyloid fibril formation by an 11-residue peptide derived from human apolipoprotein C-II (apoC-II(60-70)). In contrast, sedimentation equilibrium analysis of these peptide-lipid mixtures indicated the presence of soluble oligomeric complexes. To acquire insight into the atomic level influences of these lipids on the initial stages of aggregation of the peptide, we performed molecular dynamics (MD) simulations coupled with umbrella sampling to determine dimerization free energies of a number of beta-stranded and random coil dimer complexes, both in the presence and absence of lipids. The simulations indicate that, in contrast to their inhibitory effects on fibril formation, short-chain phospholipids promote the formation and stabilization of dimers by enhancing intersubunit hydrophobic interactions. On the basis of these experimental and computational results, we propose that peptide-bound lipids can inhibit amyloid fibril formation by trapping of dimers and other oligomeric species in diverse nonfibril forming conformations, reducing their likelihood of acquiring subunit conformations prone to fibril nucleation and growth. In light of the demonstrated cytotoxicity of amyloid peptide oligomers, our results suggest that, by enhancing the stability of oligomeric peptide species, the presence of solvated lipids may contribute to the cytotoxicity of fibrillogenic proteins and peptides.

The misfolding and self-assembly of proteins into amyloid fibrils that occurs in several debilitating and age-related diseases is affected by common components of amyloid deposits, notably lipids and lipid complexes. We have examined the effect of the short-chain phospholipids, dihexanoylphosphatidylcholine (DHPC) and dihexanoylphosphatidylserine (DHPS), on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Micellar DHPC and DHPS strongly inhibited apoC-II fibril formation, whereas submicellar levels of these lipids accelerated apoC-II fibril formation to a similar degree. These results indicate that the net negative charge on DHPS, compared with the neutrally charged DHPC, is not critical for either the inhibition or activation process. We also investigated the mechanism for the submicellar, lipid-induced activation of fibril formation. Emission data for fluorescently labeled apoC-II indicated that DHPC and DHPS stimulate the early formation and accumulation of oligomeric species. Sedimentation velocity and equilibrium experiments using a new fluorescence detection system identified a discrete lipid-induced tetramer formed at low apoC-II concentrations in the absence of significant fibril formation. Seeding experiments showed that this tetramer was on the fibril-forming pathway. Fluorescence resonance energy transfer experiments established that this tetramer forms rapidly and is stabilized by submicellar, but not micellar, concentrations of DHPC and DHPS. Several recent studies show that oligomeric intermediates in amyloid fibril formation are toxic. Our results indicate that lipids promote on-pathway intermediates of apoC-II fibril assembly and that the accumulation of a discrete tetrameric intermediate depends on the molecular state of the lipid.

We have performed experimental and computational studies to investigate the influences of phospholipids, methionine oxidation and acidic pH on amyloid fibril formation by a peptide derived from human apolipoprotein C-II (apoC-II), a known component of proteinaceous atherosclerotic plaques. Fibril growth monitored by thioflavin T fluorescence revealed inhibition under lipid-rich and oxidising conditions. We subsequently performed fully-solvated atomistic molecular dynamics (MD) simulations of the peptide monomer to study its conformations under both fibril favouring (neutral and low pH) and inhibiting (lipid-rich and oxidising) conditions. Examination of the chain topology, backbone hydrogen-bonding patterns and aromatic sidechain orientations of the peptide under different conditions reveals that, while the peptide adopts similar structures under the fibril-favouring conditions, significantly different structures are obtained under fibril-disruptive conditions. Based on our results, we advance hypotheses for the roles of peptide conformation on aggregation and fibrillisation propensities.

Methionine residues are linked to the pathogenicity of several amyloid diseases; however, the mechanism of this relationship is largely unknown. These diseases are characterized, in vivo, by the accumulation of insoluble proteinaceous plaques, of which the major constituents are amyloid fibrils. In vitro, methionine oxidation has been shown to modulate fibril assembly in several well-characterized amyloid systems. Human apolipoprotein (apo) C-II contains two methionine residues (Met-9 and Met-60) and readily self-assembles in vitro to form homogeneous amyloid fibrils, thus providing a convenient system to examine the effect of methionine oxidation on amyloid fibril formation and stability. Upon oxidation of the methionine residues of apoC-II with hydrogen peroxide, fibril formation was inhibited. Oxidized apoC-II molecules did not inhibit native apoC-II assembly, indicating that the oxidized molecules had a reduced ability to interact with the growing fibrils. Single Met-Val substitutions were performed and showed that oxidation of Met-60 had a more significant inhibitory effect than oxidation of Met-9. In addition, Met-Gln substitutions designed to mimic the effect of oxidation on side chain hydrophilicity showed that a change in hydrophobicity at position 60 within the core region of the fibril had a potent inhibitory effect. The oxidation of preformed apoC-II fibrils caused their dissociation; however, mutants in which the Met-60 was substituted with a valine were protected from this peroxide-induced dissociation. This work highlights an important role for methionine in the formation of amyloid fibril structure and gives new insight into how oxidation affects the stability of mature fibrils.

Analytical ultracentrifugation (AUC) was used to characterize the size distribution and surface chemistry of quantum dots (QDs). AUC was found to be highly sensitive to nanocrystal size, resolving nanocrystal sizes that differ by a single lattice plane. Sedimentation velocity data were used to calculate the ligand packing density at the crystal surface for different sized nanocrystals. Dihydrolipoic acid poly(ethylene glycol) was found to bind between 66 and 60% of the surface cadmium atoms for CdSe nanocrystals in the 1.54-2.59 nm radius size regime. The surface ligand chemistry was found to affect QD sedimentation, with larger ligands decreasing the sedimentation rate through an increase in particle volume and increase in frictional coefficient. Finally, AUC was used to detect and analyze protein association to QDs. Addition of bovine serum albumin (BSA) to the QD sample resulted in a reduced sedimentation rate, which may be attributed to an associated frictional drag. We calculated that one to two BSA molecules bind per QD with an associated frictional ratio of 1.2.

Alzheimer's and several other diseases are characterized by the misfolding and assembly of protein subunits into amyloid fibrils. Current models propose that amyloid fibril formation proceeds via the self-association of several monomers to form a nucleus, which then elongates by the addition of monomer to form mature fibrils. We have examined the concentration-dependent kinetics of apolipoprotein C-II amyloid fibril formation and correlated this with the final size distribution of the fibrils determined by sedimentation velocity experiments. In contrast to predictions of the nucleation-elongation model, the final size distribution of the fibrils was found to be relatively independent of the starting monomer concentration. To explain these results, we extended the nucleation-elongation model to include fibril breaking and rejoining as integral parts of the amyloid fibril assembly mechanism. The system was examined under conditions that affected the stability of the mature fibrils including the effect of dilution on the free pool of monomeric apolipoprotein C-II and the time-dependent recovery of fibril size following sonication. Antibody-labelling transmission electron microscopy studies provided direct evidence for spontaneous fibril breaking and rejoining. These studies establish the importance of breaking and rejoining in amyloid fibril formation and identify prospective new therapeutic targets in the assembly pathway.

A common feature of many of the most important and prominent amyloid-forming proteins is their ability to bind lipids and lipid complexes. Lipids are ubiquitous components of disease-associated amyloid plaques and deposits in humans, yet the specific roles of lipid in the process of amyloid fibril formation are poorly understood. This study investigated the effect of phospholipids on amyloid fibril formation by human apolipoprotein (apo) C-II using phosphatidylcholine derivatives comprising acyl chains of up to 14 carbon atoms. Submicellar concentrations of short-chain phospholipids increase the rate of apoC-II fibril formation in an acyl-chain-length- and concentration-dependent fashion, while high micellar concentrations of phospholipids completely inhibited amyloid formation. At lower concentrations of soluble phospholipid complexes, fibril formation by apoC-II was only partially inhibited, and under these conditions, aggregation followed a two-phase process. Electron microscopy showed that the fibrils resulting from the second phase of aggregation were straight, cablelike, and about 13 nm wide, in contrast to the homogeneous twisted-ribbon morphology of apoC-II fibrils formed under lipid-free conditions. Seeding experiments showed that this alternative fibril structure could be templated both in the presence and in the absence of lipid complex, suggesting that the two morphologies result from distinct assembly pathways. Circular dichroism spectroscopy studies indicated that the secondary structural conformation within the straight-type and ribbon-type fibrils were distinct, further suggesting divergent assembly pathways. These studies show that phospholipid complexes can change the structural architecture of mature fibrils and generate new fibril morphologies with the potential to alter the in vivo behaviour of amyloid. Such lipid interactions may play a role in defining the structural features of fibrils formed by diverse amyloidogenic proteins.

Apolipoprotein (apo) E is a well characterized lipid-binding protein in plasma that also exists as a common nonfibrillar component of both cerebral and systemic amyloid deposits. A genetic link between a common isoform of apoE, apoE4, and the incidence of late onset Alzheimer disease has drawn considerable attention to the potential roles of apoE in amyloid-related disease. We examined the interactions of apoE with amyloid fibrils composed of apoC-II and the amyloid-beta (Abeta) peptide. Aggregates of apoE with Abeta and apoC-II are found in Alzheimer and atherosclerotic plaques, respectively. Sedimentation velocity and fibril size distribution analysis showed that apoE3 and E4 isoforms bind and noncovalently cross-link apoC-II fibrils in a similar manner. This ability to cross-link apoC-II fibrils was abolished by the dissociation of the apoE tetramer to monomers or by thrombin cleavage to yield separate N- and C-terminal domains. Preparative ultracentrifuge binding studies indicated that apoE and the isolated N- and C-terminal domains of apoE bind with submicromolar affinities to both apoC-II and Abeta fibrils. Fluorescence quenching and resonance energy transfer experiments confirmed that both domains of apoE interact with apoC-II fibrils and demonstrated that the binding of the isolated N-terminal domain of apoE to apoC-II or Abeta fibrils is accompanied by a significant conformational change with helix three of the domain moving relative to helix one. We propose a model involving the interaction of apoE with patterns of aligned residues that could explain the general ability of apoE to bind to a diverse range of amyloid fibrils.

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of "turns" in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.