Modelling amyloid fibril formation kinetics: mechanisms of nucleation and growth.

Amyloid and amyloid-like fibrils are self-assembling protein nanostructures, of interest for their robust material properties and inherent biological compatibility as well as their putative role in a number of debilitating mammalian disorders. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. As such, this area of research has attracted significant attention over the last half century. This review describes a number of different models that have been formulated to describe the kinetics of fibril assembly. We describe the macroscopic implications of mechanisms in which secondary processes such as secondary nucleation, fragmentation or branching dominate the assembly pathway, compared to mechanisms dominated by the influence of primary nucleation. We further describe how experimental data can be analysed with respect to the predictions of kinetic models.

Ultrastructural organization of amyloid fibrils by atomic force microscopy.

Atomic force microscopy has been employed to investigate the structural organization of amyloid fibrils produced in vitro from three very different polypeptide sequences. The systems investigated are a 10-residue peptide derived from the sequence of transthyretin, the 90-residue SH3 domain of bovine phosphatidylinositol-3'-kinase, and human wild-type lysozyme, a 130-residue protein containing four disulfide bridges. The results demonstrate distinct similarities between the structures formed by the different classes of fibrils despite the contrasting nature of the polypeptide species involved. SH3 and lysozyme fibrils consist typically of four protofilaments, exhibiting a left-handed twist along the fibril axis. The substructure of TTR(10-19) fibrils is not resolved by atomic force microscopy and their uniform appearance is suggestive of a regular self-association of very thin filaments. We propose that the exact number and orientation of protofilaments within amyloid fibrils is dictated by packing of the regions of the polypeptide chains that are not directly involved in formation of the cross-beta core of the fibrils. The results obtained for these proteins, none of which is directly associated with any human disease, are closely similar to those of disease-related amyloid fibrils, supporting the concept that amyloid is a generic structure of polypeptide chains. The detailed architecture of an individual fibril, however, depends on the manner in which the protofilaments assemble into the fibrillar structure, which in turn is dependent on the sequence of the polypeptide and the conditions under which the fibril is formed.

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

Chemical dissection and reassembly of amyloid fibrils formed by a peptide fragment of transthyretin.

We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.

Apolipoprotein C-II39-62 activates lipoprotein lipase by direct lipid-independent binding.

Apolipoprotein C-II (apoC-II) is an exchangeable plasma apolipoprotein and an endogenous activator of lipoprotein lipase (LpL). Genetic deficiencies of apoC-II and overexpression of apoC-II in transgenic mice are both associated with severe hyperlipidemia, indicating a complex role for apoC-II in the regulation of blood lipid levels. ApoC-II exerts no effect on the activity of LpL for soluble substrates, suggesting that activation occurs via the formation of a lipid-bound complex. We have synthesized a peptide corresponding to amino acid residues 39-62 of mature human apoC-II. This peptide does not bind to model lipid surfaces but retains the ability to activate LpL. Conjugation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) to the N-terminal alpha-amino group of apoC-II39-62 facilitated determination of the affinity of the peptide for LpL using fluorescence anisotropy measurements. The dissociation constant describing this interaction was 0.23 microM, and was unchanged when LpL was lipid-bound. Competitive binding studies showed that apoC-II39-62 and full-length apoC-II exhibited the same affinity for LpL in aqueous solution, whereas the affinity for full-length apoC-II was increased at least 1 order of magnitude in the presence of lipid. We suggest that while the binding of apoC-II to the lipid surface promotes the formation of a high-affinity complex of apoC-II and LpL, activation occurs via direct helix-helix interactions between apoC-II39-62 and the loop covering the active site of LpL.

Mass spectrometry to characterize the binding of a peptide to a lipid surface.

The binding of an amphipathic alpha-helical peptide to small unilamellar lipid vesicles has been examined using chemical derivitization and mass spectrometry. The peptide is derived from the sequence of human apolipoprotein C-II (apoC-II), the protein activator of lipoprotein lipase (LpL). ApoC-II(19-39) forms approximately 60% alpha-helix upon binding to model egg yolk phosphatidylcholine small unilamellar vesicles. Measurement of the affinity of the peptide for lipid by spectrophotometric methods is complicated by the contribution of scattered light to optical signals. Instead, we characterize the binding event using the differential labeling of lysine residues by the lipid- and aqueous-phase cross-linkers, disuccinimidyl suberate (DSS) and bis(sulfosuccinimidyl) suberate (BS(3)), respectively. In aqueous solution, the three lysine residues of the peptide are accessible to both cross-linkers. In the presence of lipid, the C-terminal lysine residue becomes inaccessible to the lipid-phase cross-linker DSS, but remains accessible to the aqueous-phase cross-linker, BS(3). We use mass spectrometry to characterize this binding event and to derive a dissociation constant for the interaction (K(d) = 5 microM). We also provide evidence for the formation of dimeric cross-linked peptide when high densities of peptide are bound to the lipid surface.

Helix-helix association of a lipid-bound amphipathic alpha-helix derived from apolipoprotein C-II.

The interaction of a peptide derived from the sequence of apolipoprotein C-II (apoC-II) with a model lipid surface has been investigated by fluorescence spectroscopy. ApoC-II19-39, labeled at the N-terminus with 7-nitrobenz-2-oxa-1,3-diazole (NBD), bound to small unilamellar vesicles of phosphatidylcholine with a dissociation constant of 6 microM. The lipid-bound NBD-labeled peptide exhibited a red-edge excitation shift in its emission maximum and anisotropy, consistent with insertion of the probe into the motionally restricted, polar environment provided by the bilayer interface. The small Stokes shift of the NBD fluorophore permits electronic energy homotransfer between peptides on the lipid surface and results in depolarization of the NBD emission. At high surface densities of lipid-bound peptide, the anisotropy of the NBD probe was 33% lower than in corresponding samples in which electronic energy homotransfer was prevented by the addition of an unlabeled peptide. The efficiency of energy transfer between probes was not consistent with a random distribution of peptides on the lipid surface, indicating instead the self-association of lipid-bound apoC-II19-39. We propose that the role of this sequence in apoC-II is not only to mediate binding of protein to a lipid surface, but also to stabilize the lipoprotein complexes by associating with other amphipathic helices within apoC-II and with other apolipoproteins.

Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.

Trifluoroethanol induces the self-association of specific amphipathic peptides.

We have examined the effect of trifluoroethanol (TFE) on the solution behaviour of three amphipathic peptides. One of the peptides, containing three heptad repeat units (Ac-YS-(AKEAAKE)3GAR-NH2), remained monomeric under conditions where TFE induced a two-state transition from a random coil to an alpha-helix. In contrast, the TFE-induced alpha-helical formation of two peptides derived from human apolipoproteins C-II and E was accompanied by the formation of discrete dimers and trimers, respectively. The apolipoprotein C-II peptide further aggregated to form beta-sheet at higher concentrations of TFE (50% v/v). The results suggest a class of peptides capable of discrete self-association in the presence of cosolvents which favour intramolecular hydrogen bonding.

Interaction of lipoprotein lipase with homogeneous lipid emulsions.

The central function of lipoprotein lipase (LpL) is to hydrolyze triacylglycerols in chylomicrons and very low density lipoproteins. We have examined the binding of purified milk lipoprotein lipase to homogeneous synthetic lipid emulsions. Emulsions composed of either naturally occurring ester-linked lipids or the non-hydrolyzable ether analogues were prepared by sonication and pressure extrusion, and fractionated by sucrose density gradient centrifugation. Flotation analysis using the analytical ultracentrifuge indicated that the individual fractions were relatively homogeneous with respect to size with flotation coefficients and molecular weights for the separated fractions ranging from 100 to 1100 S and 5.2 x 10(7) to 6.0 x 10(8), respectively. Purified milk lipoprotein lipase bound with high affinity and in a saturable manner to emulsions prepared from the non-hydrolyzable ether-linked lipid analogues of 1-oleoyl, 2-palmitoyl phosphatidylcholine and triolein. At low concentrations of LpL, the enzyme caused aggregation of the emulsion particles by interparticle cross-linking. At higher LpL concentrations, the flotation coefficient of the emulsions decreased significantly with a concomitant increase in particle density. At saturation, the number of LpL monomers bound to lipid particles of radii 67, 75, and 79 nm was 1315, 1449, and 1466, respectively. The results demonstrate close packing of LpL on the lipid surface and are consistent with there being little disruption to the overall structure of the emulsion particle.