Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

Ethanol induced the formation of β‐sheet and amyloid‐like fibrils by surfactant‐like peptide A6K

Self‐assembly of natural or designed peptides into fibrillar structures based on β‐sheet conformation is a ubiquitous and important phenomenon. Recently, organic solvents have been reported to play inductive roles in the process of conformational change and fibrillization of some proteins and peptides. In this study, we report the change of secondary structure and self‐assembling behavior of the surfactant‐like peptide A6K at different ethanol concentrations in water. Circular dichroism indicated that ethanol could induce a gradual conformational change of A6K from unordered secondary structure to β‐sheet depending upon the ethanol concentration. Dynamic light scattering and atomic force microscopy revealed that with an increase of ethanol concentration the nanostructure formed by A6K was transformed from nanosphere/string‐of‐beads to long and smooth fibrils. Furthermore, Congo red staining/binding and thioflavin‐T binding experiments showed that with increased ethanol concentration, the fibrils formed by A6K exhibited stronger amyloid fibril features. These results reveal the ability of ethanol to promote β‐sheet conformation and fibrillization of the surfactant‐like peptide, a fact that may be useful for both designing self‐assembling peptide nanomaterials and clarifying the molecular mechanism behind the formation of amyloid fibrils. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Exploring the free energy landscape of a model β‐hairpin peptide and its isoform

Secondary structural transitions from α‐helix to β‐sheet conformations are observed in several misfolding diseases including Alzheimer's and Parkinson's. Determining factors contributing favorably to the formation of each of these secondary structures is therefore essential to better understand these disease states. β‐hairpin peptides form basic components of anti‐parallel β‐sheets and are suitable model systems for characterizing the fundamental forces stabilizing β‐sheets in fibrillar structures. In this study, we explore the free energy landscape of the model β‐hairpin peptide GB1 and its E2 isoform that preferentially adopts α‐helical conformations at ambient conditions. Umbrella sampling simulations using all‐atom models and explicit solvent are performed over a large range of end‐to‐end distances. Our results show the strong preference of GB1 and the E2 isoform for β‐hairpin and α‐helical conformations, respectively, consistent with previous studies. We show that the unfolded states of GB1 are largely populated by misfolded β‐hairpin structures which differ from each other in the position of the β‐turn. We discuss the energetic factors contributing favorably to the formation of α‐helix and β‐hairpin conformations in these peptides and highlight the energetic role of hydrogen bonds and non‐bonded interactions. Proteins 2014; 82:2394–2402. © 2014 Wiley Periodicals, Inc.     

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid‐β peptide 1‐42 but not amylin and insulin fibrils can grow under quiescent conditions

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer's amyloid‐β (Aβ) 1‐40 and 1‐42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ₄₀ substantially decreased, whereas the fibrillization of Aβ₄₂ peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Effect of external electrostatic field on the stability of β sheet structures

To explore the effect of an external electrostatic field (EEF) on the stability of protein conformations, the molecular dynamic modeling approach was applied to evaluate the effect of an EEF along the x or y direction on a water cluster containing a parallel or antiparallel β sheet structure. The β sheet structure contained two strands with a (Gly)₃ sequence separated by a distance d along the x direction. The mean forces between the two strands along the x direction were computed from the trajectories of molecular dynamics simulations. In the absence of the EEF, the forces between the two strands in vacuum were repulsive and attractive in the parallel and antiparallel β sheet structures, respectively. In contrast, the mean forces between the two strands in water were attractive in both the parallel and antiparallel β sheet structures. This is because the electric interactions between the two strands were shielded by water, and the hydrophobic effect dominated the interaction between the two strands. When an EEF >50 MV/cm was applied to the water cluster, the attractive force between the two strands in the parallel and antiparallel β sheet structures decreased and increased, respectively. Further, the binding affinity between the two strands in the parallel and antiparallel β sheet structures also decreased and increased, respectively. This is because the large EEF leads to dielectric saturation, and consequently reduces the effects of the dielectric shielding and hydrophobic interactions. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 861–870, 2014.     

Morphology of self‐assembled structures formed by short peptides from the amyloidogenic protein tau depends on the solvent in which the peptides are dissolved

The aggregation behavior of peptides Ac‐VQIVYK‐amide (AcPHF6) and Ac‐QIVYK‐amide (AcPHF5) from the amyloidogenic protein tau was examined by atomic force microscopy (AFM) and fluorescence microscopy. Although AcPHF5 did not show enhancement of thioflavin T (ThT) fluorescence in aqueous buffer, distinct aggregates were discernible when peptide was dissolved in organic solvents such as methanol (MeOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) dried on mica and examined by AFM. Self‐association was evident even though the peptide did not have the propensity to form secondary structures in the organic solvents. In dried films, the peptide adopts predominantly β‐conformation which results in the formation of distinct aggregates. ThT fluorescence spectra and fluorescence images indicate the formation of fibrils when AcPHF6 solutions in organic solvents were diluted into buffer. AcPHF6 had the ability to organize into fibrillar structures when AFM samples were prepared from peptide dissolved in MeOH, TFE, HFIP, and also when diluted into buffer. AcPHF6 showed propensity for β‐structure in aqueous buffer. In MeOH and TFE, AcPHF6 showed helical and β‐structure. Morphology of the fibrils was dependent on peptide conformation in the organic solvents. The structures observed for AcPHF6 are formed rapidly and long incubation periods in the solvents are not necessary. The structures with varying morphologies observed for AcPHF5 and AcPHF6 appear to be mediated by surfaces such as mica and the organic solvents used for dissolution of the peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

Computer simulation study of amyloid fibril formation by palindromic sequences in prion peptides

We simulate the aggregation of large systems containing palindromic peptides from the Syrian hamster prion protein SHaPrP 113-120 (AGAAAAGA) and the mouse prion protein MoPrP 111-120 (VAGAAAAGAV) and eight sequence variations: GAAAAAAG, (AG)(4) , A8, GAAAGAAA, A10, V10, GAVAAAAVAG, and VAVAAAAVAV The first two peptides are thought to act as the Velcro that holds the parent prion proteins together in amyloid structures and can form fibrils themselves. Kinetic events along the fibrillization pathway influence the types of structures that occur and variations in the sequence affect aggregation kinetics and fibrillar structure. Discontinuous molecular dynamics simulations using the PRIME20 force field are performed on systems containing 48 peptides starting from a random coil configuration. Depending on the sequence, fibrillar structures form spontaneously over a range of temperatures, below which amorphous aggregates form and above which no aggregation occurs. AGAAAAGA forms well organized fibrillar structures whereas VAGAAAAGAV forms less well organized structures that are partially fibrillar and partially amorphous. The degree of order in the fibrillar structure stems in part from the types of kinetic events leading up to its formation, with AGAAAAGA forming less amorphous structures early in the simulation than VAGAAAAGAV. The ability to form fibrils increases as the chain length and the length of the stretch of hydrophobic residues increase. However as the hydrophobicity of the sequence increases, the ability to form well-ordered structures decreases. Thus, longer hydrophobic sequences form slightly disordered aggregates that are partially fibrillar and partially amorphous. Subtle changes in sequence result in slightly different fibril structures.     

Impact of sequence on the molecular assembly of short amyloid peptides

The goal of this work is to understand how the sequence of a protein affects the likelihood that it will form an amyloid fibril and the kinetics along the fibrillization pathway. The focus is on very short fragments of amyloid proteins since these play a role in the fibrillization of the parent protein and can form fibrils themselves. Discontinuous molecular dynamics simulations using the PRIME20 force field were performed of the aggregation of 48‐peptide systems containing SNQNNF ( PrP (170–175 )), SSTSAA (RNaseA(15–20)), MVGGVV (Aβ(35–40)), GGVVIA (Aβ(37–42)), and MVGGVVIA (Aβ(35–42)). In our simulations SNQQNF, SSTTSAA, and MVGGVV form large numbers of fibrillar structures spontaneously (as in experiment). GGVVIA forms β‐sheets that do not stack into fibrils (unlike experiment). The combination sequence MVGGVVIA forms less fibrils than MVGGVV, hindered by the presence of the hydrophobic residues at the C‐terminal. Analysis of the simulation kinetics and energetics reveals why MVGGVV forms fibrils and GGVVIA does not, and why adding I and A to MVGGVVIA reduces fibrillization and enhances amorphous aggregation into oligomeric structures. The latter helps explain why Aβ(1–42) assembles into more complex oligomers than Aβ(1–40), a consequence of which is that it is more strongly associated with Alzheimer's disease. Proteins 2014; 82:1469–1483. © 2014 Wiley Periodicals, Inc.     

β‐sheet assembly in amyloidogenic glutamic acid nanostructures: Insights from X‐ray scattering and infrared nanospectroscopy

Glutamic acid–rich peptides are crucial to a variety of biological processes, including glutamatergic neurotransmission and immunological defense. Glutamic acid sequences often exhibit unusual organization into β₂‐type sheets, where bifurcated H bonds formed between glutamic acid side chains and NH in amide bonds on adjacent β‐strands play a paramount role for stabilizing the molecular assembly. Herein, we investigate the self‐assembly and supramolecular structure of simplified models consisting of alternating glutamic acid/phenylalanine residues. Small‐angle X‐ray scattering and atomic force microscopy show that the aggregation pathway is characterized by the formation of small oligomers, followed by coalescence into nanofibrils and nanotapes. Amyloidogenic features are further demonstrated through fiber X‐ray diffraction, which reveal molecular packing according to cross‐β patterns, where β‐strands appear perpendicularly oriented to the long axis of nanofibrils and nanotapes. Nanoscale infrared spectroscopy from individual nanoparticles on dried samples shows a remarkable decrease of β₂‐sheet content, accompanied by growth of standard β‐sheet fractions, indicating a β₂‐to‐β₁ transition as a consequence of the release of solvent from the interstices of peptide assemblies. Our findings highlight the key role played by water molecules in mediating H‐bond formation in β₂‐sheets commonly found in amyloidogenic glutamic acid–rich aggregates.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.     

Molecular mechanism of β-sheet self-organization at water-hydrophobic interfaces

The capacity to form β‐sheet structure and to self‐organize into amyloid aggregates is a property shared by many proteins. Severe neurodegenerative pathologies such as Alzheimer's disease are thought to involve the interaction of amyloidogenic protein oligomers with neuronal membranes. To understand the experimentally observed catalysis of amyloid formation by lipid membranes and other water‐hydrophobic interfaces, we examine the physico‐chemical basis of peptide adsorption and aggregation in a model membrane using atomistic molecular simulations. Blocked octapeptides with simple, repetitive sequences, (Gly‐Ala)₄, and (Gly‐Val)₄, are used as models of β‐sheet‐forming polypeptide chains found in the core of amyloid fibrils. In the presence of an n‐octane phase mimicking the core of lipid membranes, the peptides spontaneously partition at the octane‐water interface. The adsorption of nonpolar sidechains displaces the peptides' conformational equilibrium from a heterogeneous ensemble characterized by a high degree of structural disorder toward a more ordered ensemble favoring β‐hairpins and elongated β‐strands. At the interface, peptides spontaneously aggregate and rapidly evolve β‐sheet structure on a 10 to 100 ns time scale, while aqueous aggregates remain amorphous. Catalysis of β‐sheet formation results from the combination of the hydrophobic effect and of reduced conformational entropy of the polypeptide chain. While the former drives interfacial partition and displaces the conformational equilibrium of monomeric peptides, the planar interface further facilitates β‐sheet organization by increasing peptide concentration and reducing the dimensionality of self‐assembly from three to two. These findings suggest a general mechanism for the formation of β‐sheets on the surface of globular proteins and for amyloid self‐organization at hydrophobic interfaces. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

The protonation state of histidine 111 regulates the aggregation of the evolutionary most conserved region of the human prion protein

In a group of neurodegenerative diseases, collectively termed transmissible spongiform encephalopathies, the prion protein aggregates into β‐sheet rich amyloid‐like deposits. Because amyloid structure has been connected to different prion strains and cellular toxicity, it is important to obtain insight into the structural properties of prion fibrils. Using a combination of solution NMR spectroscopy, thioflavin‐T fluorescence and electron microscopy we here show that within amyloid fibrils of a peptide containing residues 108–143 of the human prion protein [humPrP (108–143)]—the evolutionary most conserved part of the prion protein ‐ residue H111 and S135 are in close spatial proximity and their interaction is critical for fibrillization. We further show that residues H111 and H140 share the same microenvironment in the unfolded, monomeric state of the peptide, but not in the fibrillar form. While protonation of H140 has little influence on fibrillization of humPrP (108–143), a positive charge at position 111 blocks the conformational change, which is necessary for amyloid formation of humPrP (108–143). Our study thus highlights the importance of protonation of histidine residues for protein aggregation and suggests point mutations to probe the structure of infectious prion particles.     

PackHelix: a tool for helix-sheet packing during protein structure prediction

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α‐helices and/or β‐strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β‐sheet in a protein, there has been little progress on generating native‐like packings of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β‐sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ₂ motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. Proteins 2011; Published 2011 Wiley‐Liss, Inc.     

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer''s disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.     

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two‐dimensional homonuclear NMR

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific ¹H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999