Partially folded intermediates as critical precursors of light chain amyloid fibrils and amorphous aggregates

Light chain, or AL, amyloidosis is a pathological condition arising from systemic extracellular deposition of monoclonal immunoglobulin light chain variable domains in the form of insoluble amyloid fibrils, especially in the kidneys. Substantial evidence suggests that amyloid fibril formation from native proteins occurs via a conformational change leading to a partially folded intermediate conformation, whose subsequent association is a key step in fibrillation. In the present investigation, we have examined the properties of a recombinant amyloidogenic light chain variable domain, SMA, to determine whether partially folded intermediates can be detected and correlated with aggregation. The results from spectroscopic and hydrodynamic measurements, including far- and near-UV circular dichroism, FTIR, NMR, and intrinsic tryptophan fluorescence and small-angle X-ray scattering, reveal the build-up of two partially folded intermediate conformational states as the pH is decreased (low pH destabilized the protein and accelerated the kinetics of aggregation). A relatively nativelike intermediate, I(N), was observed between pH 4 and 6, with little loss of secondary structure, but with significant tertiary structure changes and enhanced ANS binding, indicating exposed hydrophobic surfaces. At pH below 3, we observed a relatively unfolded, but compact, intermediate, I(U), which was characterized by decreased tertiary and secondary structure. The I(U) intermediate readily forms amyloid fibrils, whereas I(N) preferentially leads to amorphous aggregates. Except at pH 2, where negligible amorphous aggregate is formed, the amorphous aggregates formed significantly more rapidly than the fibrils. This is the first indication that different partially folded intermediates may be responsible for different aggregation pathways (amorphous and fibrillar). The data support the hypothesis that amyloid fibril formation involves the ordered self-assembly of partially folded species that are critical soluble precursors of fibrils.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Conformational conversion during amyloid formation at atomic resolution

Numerous studies of amyloid assembly have indicated that partially folded protein species are responsible for initiating aggregation. Despite their importance, the structural and dynamic features of amyloidogenic intermediates and the molecular details of how they cause aggregation remain elusive. Here, we use ΔN6, a truncation variant of the naturally amyloidogenic protein β(2)-microglobulin (β(2)m), to determine the solution structure of a nonnative amyloidogenic intermediate at high resolution. The structure of ΔN6 reveals a major repacking of the hydrophobic core to accommodate the nonnative peptidyl-prolyl trans-isomer at Pro32. These structural changes, together with a concomitant pH-dependent enhancement in backbone dynamics on a microsecond-millisecond timescale, give rise to a rare conformer with increased amyloidogenic potential. We further reveal that catalytic amounts of ΔN6 are competent to convert nonamyloidogenic human wild-type β(2)m (Hβ(2)m) into a rare amyloidogenic conformation and provide structural evidence for the mechanism by which this conformational conversion occurs.     

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.     

Review: immunoglobulin light chain amyloidosis--the archetype of structural and pathogenic variability

AL amyloidosis is caused by deposition in target tissue of amyloid fibrils constituted by monoclonal immunoglobulin light chains. The amyloidogenic plasma cells derive from a transformed memory B cell that can be identified by anti-idiotype monoclonal antibodies. Comparison of the primary structures of amyloidogenic and nonamyloidogenic light chains does not show any common structural motif in the amyloidogenic variants but reveals peculiar replacements which can destabilize the folding state. Reduced folding stability now appears to be a unifying property of amyloidogenic light chains. The tendency of these proteins to populate a partially unfolded intermediate state is a key event in the self-association that progresses to the formation of oligomers and fibrils. The mechanism of organ damage caused by AL amyloid deposition is not known, but clinical findings suggest that the process of amyloid fibril formation itself exerts tissue toxic effects independently of the amount of amyloid deposited. Since the disease is caused by the neoplastic expansion of the plasma cell population synthesizing the amyloidogenic light chains, the clone represents the prime therapeutic target of conventional chemotherapy and experimental immunotherapy. In common with other types of amyloidosis the therapeutic strategy can take advantage of drugs able to improve the reabsorption of the amyloid deposits or able to bind and stabilize the light chain in the native-like folded state.     

Structural properties of an amyloid precursor of beta(2)-microglobulin

The population of one or more partially folded states has been proposed as a critical initial step in amyloid formation for several proteins. Here we use equilibrium denaturation measured by (1)H-(15)N NMR to determine the conformational properties of an amyloidogenic intermediate of human beta(2)-microglobulin (beta(2)m) formed at low pH. The data show that this amyloid precursor is a noncooperatively stabilized ensemble that retains stable structure in five of the seven beta-strands that comprise the native fold. The amyloid precursors of beta(2)m and transthyretin have similar properties despite having structurally unrelated native folds. The data offer a rationale as to why these proteins are both amyloidogenic at low pH and suggest that amyloidosis of these and other proteins may involve ordered assembly from a precursor with similar conformational features.     

Amyloid fibril formation by macrophage migration inhibitory factor

We demonstrate herein that human macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine expressed in the brain and not previously considered to be amyloidogenic, forms amyloid fibrils similar to those derived from the disease associated amyloidogenic proteins beta-amyloid and alpha-synuclein. Acid denaturing conditions were found to readily induce MIF to undergo amyloid fibril formation. MIF aggregates to form amyloid-like structures with a morphology that is highly dependent on pH. The mechanism of MIF amyloid formation was probed by electron microscopy, turbidity, Thioflavin T binding, circular dichroism spectroscopy, and analytical ultracentrifugation. The fibrillar structures formed by MIF bind Congo red and exhibit the characteristic green birefringence under polarized light. These results are consistent with the notion that amyloid fibril formation is not an exclusive property of a select group of amyloidogenic proteins, and contribute to a better understanding of the factors which govern protein conformational changes and amyloid fibril formation in vivo.     

Mutations can cause light chains to be too stable or too unstable to form amyloid fibrils

Light chain (AL) amyloidosis is an incurable human disease, where the amyloid precursor is a misfolding‐prone immunoglobulin light‐chain. Here, we identify the role of somatic mutations in the structure, stability and in vitro fibril formation for an amyloidogenic AL‐12 protein by restoring four nonconservative mutations to their germline (wild‐type) sequence. The single restorative mutations do not affect significantly the native structure, the unfolding pathway, and the reversibility of the protein. However, certain mutations either decrease (H32Y and H70D) or increase (R65S and Q96Y) the protein thermal stability. Interestingly, the most and the least stable mutants, Q96Y and H32Y, do not form amyloid fibrils under physiological conditions. Thus, Q96 and H32 are key residues for AL‐12 stability and fibril formation and restoring them to the wild‐type residues preclude amyloid formation. The mutants whose equilibrium is shifted to either the native or unfolded states barely sample transient partially folded states, and therefore do not form fibrils. These results agree with previous observations by our laboratory and others that amyloid formation occurs because of the sampling of partially folded states found within the unfolding transition (Blancas‐Mejia and Ramirez‐Alvarado, Ann Rev Biochem 2013;82:745–774). Here we provide a new insight on the AL amyloidosis mechanism by demonstrating that AL‐12 does not follow the established thermodynamic hypothesis of amyloid formation. In this hypothesis, thermodynamically unstable proteins are more prone to amyloid formation. Here we show that within a thermal stability range, the most stable protein in this study is the most amyloidogenic protein.     

Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation

Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG _D = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG _D = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.     

The role of lipid-protein interactions in amyloid-type protein fibril formation

Structural transition of polypeptide chains into the beta-sheet state followed by amyloid fibril formation is the key characteristic of a number of the so-called conformational diseases. The multistep process of protein fibrillization can be modulated by a variety of factors, in particular by lipid-protein interactions. A wealth of experimental evidence provides support to the notion that amyloid fibril assembly and the toxicity of pre-fibrillar aggregates are closely related and are both intimately membrane associated phenomena. The present review summarizes the principal factors responsible for the enhancement of fibril formation in a membrane environment, viz. (i) structural transformation of polypeptide chain into a partially folded conformation, (ii) increase of the local concentration of a protein upon its membrane binding, (iii) aggregation-favoring orientation of the bound protein, and (iv) variation in the depth of bilayer penetration affecting the nucleation propensity of the membrane associated protein. The molecular mechanisms of membrane-mediated protein fibrillization are discussed. Importantly, the toxicity of lipid-induced pre-fibrillar aggregates is likely to have presented a very strong negative selection pressure in the evolution of amino acid sequences.     

Induction of protein conformational change in mouse senile amyloidosis

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest. 81, 493-499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.     

Compact conformations of α-synuclein induced by alcohols and copper

The intrinsically disordered protein α-synuclein aggregates into amyloid fibrils, a process known to be implicated in several neurodegenerative states. Partially folded forms of the protein are thought to trigger the aggregation process. Here, α-synuclein conformers are characterized by analysis of the charge-state distributions observed in electrospray-ionization mass spectrometry under negative-ion mode. It is found that, even at neutral pH, a small fraction of the molecular population is in a compact conformation. Several distinct partially folded forms are then identified under conditions that promote α-synuclein aggregation, such as solutions of simple and fluorinated alcohols. Specific intermediates accumulate at increasing concentrations of ethanol, hexafluoro-2-propanol, and trifluoroethanol. Finally, extensive folding induced by Cu(2+) binding is revealed by titrations in the presence of Cu(2+)-glycine. The data confirm the existence of a single, high-affinity binding site for Cu(2+). Because accumulation of this partially folded form correlates with enhancement of fibrillation kinetics, it is likely to represent an amyloidogenic intermediate in α-synuclein conformational transitions.     

Elongation in a beta-structure promotes amyloid-like fibril formation of human lysozyme

To understand the mechanism of amyloid fibril formation of a protein, we examined wild-type and three mutant human lysozymes containing both amyloidogenic and non-amyloidogenic proteins: I56T (amyloidogenic); EAEA, which has four additional residues (Glu-Ala-Glu-Ala-) at the N-terminus located on a beta-structure; and EAEA-I56T, which is an I56T mutant of EAEA. All formed amyloid-like fibrils through an in the increase contents of alpha-helix with increasing concentration of ethanol. The order of propensity for amyloid-like fibril formation in highly concentrated ethanol solution is EAEA-I56T > EAEA > I56T > wild-type. This order is almost the reverse of the order of conformational stability of these proteins, wild-type > EAEA > I56T > EAEA-I56T. The important views in this work are as follows. (i) Artificially modified proteins formed amyloid fibrils in vitro. This means that amyloid formation is a generic property of polypeptide chains. (ii) The amyloidogenic mutation Ile56 to Thr caused the destabilization and promoted fibril formation in the wild-type and EAEA human lysozymes, indicating that instability facilitates amyloid formation. (iii) The mutant protein EAEA human lysozyme had higher propensity for fibril formation than the amyloidogenic mutant protein, indicating that amyloid formation is controlled not only by stability but also by other factors. In this case, appending polypeptide chains to a beta-structure accelerated amyloid formation.     

Heparin induces harmless fibril formation in amyloidogenic W7FW14F apomyoglobin and amyloid aggregation in wild-type protein in vitro

Glycosaminoglycans (GAGs) are frequently associated with amyloid deposits in most amyloid diseases, and there is evidence to support their active role in amyloid fibril formation. The purpose of this study was to obtain structural insight into GAG-protein interactions and to better elucidate the molecular mechanism underlying the effect of GAGs on the amyloid aggregation process and on the related cytotoxicity. To this aim, using Fourier transform infrared and circular diochroism spectroscopy, electron microscopy and thioflavin fluorescence dye we examined the effect of heparin and other GAGs on the fibrillogenesis and cytotoxicity of aggregates formed by the amyloidogenic W7FW14 apomyoglobin mutant. Although this protein is unrelated to human disease, it is a suitable model for in vitro studies because it forms amyloid-like fibrils under physiological conditions of pH and temperature. Heparin strongly stimulated aggregation into amyloid fibrils, thereby abolishing the lag-phase normally detected following the kinetics of the process, and increasing the yield of fibrils. Moreover, the protein aggregates were harmless when assayed for cytotoxicity in vitro. Neutral or positive compounds did not affect the aggregation rate, and the early aggregates were highly cytotoxic. The surprising result that heparin induced amyloid fibril formation in wild-type apomyoglobin and in the partially folded intermediate state of the mutant, i.e., proteins that normally do not show any tendency to aggregate, suggested that the interaction of heparin with apomyoglobin is highly specific because of the presence, in protein turn regions, of consensus sequences consisting of alternating basic and non-basic residues that are capable of binding heparin molecules. Our data suggest that GAGs play a dual role in amyloidosis, namely, they promote beneficial fibril formation, but they also function as pathological chaperones by inducing amyloid aggregation.     

Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants

Amyloid fibril formation and deposition is a common feature of a wide range of fatal diseases including spongiform encephalopathies, Alzheimer's disease, and familial amyloidotic polyneuropathies (FAP), among many others. In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein. Recently, we showed that transthyretin in solution may undergo dissociation to a non-native monomer, even under close to physiological conditions of temperature, pH, ionic strength, and protein concentration. We also showed that this non-native monomer is a compact structure, does not behave as a molten globule, and may lead to the formation of partially unfolded monomeric species and high molecular mass soluble aggregates (Quintas, A., Saraiva, M. J. M., and Brito, R. M. M. (1999) J. Biol. Chem. 274, 32943-32949). Here, based on aging experiments of tetrameric TTR and chemically induced protein unfolding experiments of the non-native monomeric forms, we show that tetramer dissociation and partial unfolding of the monomer precedes amyloid fibril formation. We also show that TTR variants with the least thermodynamically stable non-native monomer produce the largest amount of partially unfolded monomeric species and soluble aggregates under conditions that are close to physiological. Additionally, the soluble aggregates formed by the amyloidogenic TTR variants showed morphological and thioflavin-T fluorescence properties characteristic of amyloid. These results allowed us to conclude that amyloid fibril formation by some TTR variants might be triggered by tetramer dissociation to a compact non-native monomer with low conformational stability, which originates partially unfolded monomeric species with a high tendency for ordered aggregation into amyloid fibrils. Thus, partial unfolding and conformational fluctuations of molecular species with marginal thermodynamic stability may play a crucial role on amyloid formation in vivo.     

Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.

Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential Effects on Light Chain Amyloid Formation Depend on Mutations and Type of Glycosaminoglycans

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.     

Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis

Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state.     

Detecting the inter-peptide arrangement and maturation process of transthyretin (105-115) amyloid fibril using a FRET pair with short Förster distance

Transthyretin (TTR) is an amyloidogenic protein involved in many mental diseases. The peptide derived from TTR (105-115) has been widely studied as a model peptide for understanding the mechanism of amyloid fibril formation. However, the detailed arrangement of this peptide in amyloid fibril is still unclear. We have studied the amyloid fibril formation process of TTR (105-115) by introducing a pair of FRET probes into the peptide with a dansyl group at the N-terminal and a tryptophan residue at the C-terminal. Our experiment demonstrated that the strands of TTR (105-115) in the same beta-sheet may be parallel and the mating sheets may be anti-parallel to each other in the amyloid fibril. The kinetics followed by FRET and EM indicated for a possible intermediate state and the distance between sheets became shorter when the intermediate amyloid fibril turns into a more matured form.     

Amyloidogenic Mutation Promotes Fibril Formation of the N-terminal Apolipoprotein A-I on Lipid Membranes

The N-terminal amino acid 1–83 fragment of apolipoprotein A-I (apoA-I) has a strong propensity to form amyloid fibrils at physiological neutral pH. Because apoA-I has an ability to bind to lipid membranes, we examined the effects of the lipid environment on fibril-forming properties of the N-terminal fragment of apoA-I variants. Thioflavin T fluorescence assay as well as fluorescence and transmission microscopies revealed that upon lipid binding, fibril formation by apoA-I 1–83 is strongly inhibited, whereas the G26R mutant still retains the ability to form fibrils. Such distinct effects of lipid binding on fibril formation were also observed for the amyloidogenic prone region-containing peptides, apoA-I 8–33 and 8–33/G26R. This amyloidogenic region shifts from random coil to α-helical structure upon lipid binding. The G26R mutation appears to prevent this helix transition because lower helical propensity and more solvent-exposed conformation of the G26R variant upon lipid binding were observed in the apoA-I 1–83 fragment and 8–33 peptide. With a partially α-helical conformation induced by the presence of 2,2,2-trifluoroethanol, fibril formation by apoA-I 1–83 was strongly inhibited, whereas the G26R variant can form amyloid fibrils. These findings suggest a new possible pathway for amyloid fibril formation by the N-terminal fragment of apoA-I variants: the amyloidogenic mutations partially destabilize the α-helical structure formed upon association with lipid membranes, resulting in physiologically relevant conformations that allow fibril formation.