Insight into the structure of amyloid fibrils from the analysis of globular proteins

The conversion from soluble states into cross-β fibrillar aggregates is a property shared by many different proteins and peptides and was hence conjectured to be a generic feature of polypeptide chains. Increasing evidence is now accumulating that such fibrillar assemblies are generally characterized by a parallel in-register alignment of β-strands contributed by distinct protein molecules. Here we assume a universal mechanism is responsible for β-structure formation and deduce sequence-specific interaction energies between pairs of protein fragments from a statistical analysis of the native folds of globular proteins. The derived fragment–fragment interaction was implemented within a novel algorithm, prediction of amyloid structure aggregation (PASTA), to investigate the role of sequence heterogeneity in driving specific aggregation into ordered self-propagating cross-β structures. The algorithm predicts that the parallel in-register arrangement of sequence portions that participate in the fibril cross-β core is favoured in most cases. However, the antiparallel arrangement is correctly discriminated when present in fibrils formed by short peptides. The predictions of the most aggregation-prone portions of initially unfolded polypeptide chains are also in excellent agreement with available experimental observations. These results corroborate the recent hypothesis that the amyloid structure is stabilised by the same physicochemical determinants as those operating in folded proteins. They also suggest that side chain–side chain interaction across neighbouring β-strands is a key determinant of amyloid fibril formation and of their self-propagating ability.     

Nanogels prepared by self-assembly of oppositely charged globular proteins

Ovalbumin and lysozyme are two main proteins in hen egg white with the isoelectric points of 4.8 and 11, respectively. Herein we report the manufacture of stable, narrowly distributed nanogels (hydrodynamic radius about 100 nm) using a novel and convenient method: ovalbumin and lysozyme solutions were mixed at pH 5.3, the mixture solution was adjusted to pH 10.3, then subsequently stirred and heated. The nanogels were characterized using a combination of techniques. The nanogels have spherical shape and core-shell structure. The core is mainly composed of lysozyme and the shell is mainly composed of ovalbumin. The proteins in the nanogels are in denatured states and they are bound by intermolecular hydrophobic interactions, hydrogen bonds, and disulfide bonds. The charges of the nanogels can be modulated by the pH of the medium. The electrostatic repulsion of ovalbumin molecules on the nanogel surface stabilizes the nanogels in aqueous solution. The formation mechanism of the nanogels is discussed.     

Folding mechanism of all-beta globular proteins

Elucidating the mechanism for the fast folding of proteins is a challenging task. In our earlier work, we introduced the concept of "long-range order" and related it successfully to protein folding rates. In this article, we propose a new hypothesis for the folding of two-state all-beta proteins. The mechanism is based on the formation of a hydrophobic core, propagation of beta-strands, and the establishment of hydrogen bonds. Our hypothesis has been strengthened by the observation of a folding nucleus in beta-strands and the hydrogen-bonding network between residues in beta-strands. Our insights on protein folding show an excellent agreement with experimental observations.     

Structures closed into cycles in globular proteins

Different types of structures closed into cycles are widespread at all the levels of structural organization of proteins. β-Hairpins, triple-stranded β-sheets, and βαβ-units represent simple structural motifs closed into cycles by systems of hydrogen bonds. Secondary closing of these simple motifs into larger cycles by means of different superhelices, split β-hairpins, or SS-bridges results in formation of complex structural motifs such as abcd-units, φ-motifs, five- and seven-segment α/β-motifs, etc. At the level of tertiary structure many proteins and domains fold into structures closed into cylinders. Apparently, closing the motifs and domains into cycles and cylinders results in formation of more cooperative and stable structures as compared with open ones, and this may be the reason for high frequencies of occurrence of the motifs in proteins.     

How to develop globular proteins into adhesives

To make globular proteins suitable for application in adhesives, the specific bonds and interactions which shape their structure have to broken. Only then, a layer of relatively large, flexible and interwoven polymer chains, which are firmly attached to the solid surface by adsorption, can be created. Such a network layer is essential to save the adhesive bond under an applied force, because it can distribute the concentration of stresses generated at the interface into the bulk. Unfolding and swelling of a protein can be achieved by changing the solvent quality. For the globular whey protein beta-lactoglobulin, the optimal conditions for unfolding and swelling is found with 98% formic acid as a solvent. In formic acid, beta-lactoglobulin looses its amphoteric character (it is protonated, probably for approximately 20%). In addition, formic acid is less polar than water and thus a better solvent for the apolar parts of the protein. The swelling and unfolding behaviour of beta-lactoglobulin is studied by viscosity and CD-spectroscopy measurements. For the interpretation of the results we apply the Kuhn formalism that the conformation of a protein can be described in terms of a statistical chain which consists of segments of an average persistence length P. The statistical segment length P, which varies with the experimental conditions, is directly related to the adsorption energy required for a strong adhesion between coil and surface. It determines the depletion energy kT P(-2) m(-2) which must be overcome by specific attraction between side groups of the protein chain and the surface. For beta-lactoglobulin in 98% formic acid, we find a P value of approximately 2.2 nm, pointing at a relatively flexible chain. The minimum net adsorption energy kT P(-2) is then approximately 1 mJ m(-2), a relatively small value to be exceeded. Preliminary results of destructive adhesion tests on beech wood lap-shear joints reveal promising tensile strengths of approximately 2.9+/-1.1 N mm(-2), indeed.     

Natural and amyloid self-assembly of S100 proteins: structural basis of functional diversity

The S100 proteins are 10-12 kDa EF-hand proteins that act as central regulators in a multitude of cellular processes including cell survival, proliferation, differentiation and motility. Consequently, many S100 proteins are implicated and display marked changes in their expression levels in many types of cancer, neurodegenerative disorders, inflammatory and autoimmune diseases. The structure and function of S100 proteins are modulated by metal ions via Ca(2+) binding through EF-hand motifs and binding of Zn(2+) and Cu(2+) at additional sites, usually at the homodimer interfaces. Ca(2+) binding modulates S100 conformational opening and thus promotes and affects the interaction with p53, the receptor for advanced glycation endproducts and Toll-like receptor 4, among many others. Structural plasticity also occurs at the quaternary level, where several S100 proteins self-assemble into multiple oligomeric states, many being functionally relevant. Recently, we have found that the S100A8/A9 proteins are involved in amyloidogenic processes in corpora amylacea of prostate cancer patients, and undergo metal-mediated amyloid oligomerization and fibrillation in vitro. Here we review the unique chemical and structural properties of S100 proteins that underlie the conformational changes resulting in their oligomerization upon metal ion binding and ultimately in functional control. The possibility that S100 proteins have intrinsic amyloid-forming capacity is also addressed, as well as the hypothesis that amyloid self-assemblies may, under particular physiological conditions, affect the S100 functions within the cellular milieu.     

Phenomenological similarities between protein denaturation and small-molecule dissolution: Insights into the mechanism driving the thermal resistance of globular proteins

This article shows that the stability profiles of thermophilic proteins are significantly displaced toward higher temperatures as compared to those of mesophilic proteins. A similar trend characterizes the aqueous transfer of N-alkyl amides. In fact, as a general feature of transfer processes, liquid dissolution profiles are centered at temperatures higher than those of solid ones. This behavior is governed by packing contributions. A partition of the unfolding thermodynamics based on the analysis of phenomenological temperatures common to dissolution and unfolding phenomena provides a clue to understanding the mechanism of thermal stabilization. In fact, the position of stability profiles along the temperature axis does not appear to depend on solvation of internal residues. Instead, it is notably affected by solidlike components, whose progressive decrease appears to drive the heat denaturation temperature increase of most thermostable proteins. As a corollary, it is shown that there are actually two limiting mechanisms of thermal stabilization.     

Competing pathways determine fibril morphology in the self-assembly of beta2-microglobulin into amyloid

Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of beta2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.     

Native structure protects SUMO proteins from aggregation into amyloid fibrils

SUMO proteins belong to the Ubiquitin-like protein family, all sharing a common fold and a similar mechanism of conjugation to target polypeptides. SUMO is ubiquitous in all eukaryotes and participates in many crucial pathways. Native SUMO proteins are highly soluble, a property that is exploited in biotechnology. Moreover, SUMO regulates the solubility of aggregation-prone proteins in neurodegenerative disorders. Despite these properties, we show here that human SUMO1, SUMO2, and SUMO3 proteins are at risk of aggregation into amyloid structures if their native conformation is perturbed. Aggregation is mediated by specific regions, which overlap with SUMO functional interfaces, illustrating a competition between function and aggregation. Aggregation of SUMOs might have important physiological implications because disruption of the SUMO pathway is lethal in different organisms. It appears that functional constraints make it difficult to avoid the competition between productive folding and deleterious aggregation in globular proteins, even for essential polypeptides.     

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.1 MPa and 100 degrees C, respectively. In contrast to the native or unfolded protein, fibrils do not H-D exchange upon incubation at 65 degrees C. A complete deuteration of H(2)O-grown fibrils occurs when the beta-sheet structure is reassembled in a 75 wt % DMSO/D(2)O solution. Our findings suggest a densely packed environment around the amide protons involved in the intermolecular beta-sheet motive. In disagreement with the concept of "amyloid fibers as water-filled nanotubes" [Perutz, M. F., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5591-5595], elution of D(2)O-grown fibrils with H(2)O is complete, which is reflected by the vanishing of D(2)O bending vibrations at 1214 cm(-)(1). This implies the absence of "trapped water" within insulin fibrils. The rigid conformations of the native and fibrillar insulin contrast with transient intermediate states docking at the fibrils' ends. Room-temperature seeding is accompanied by an accelerated H-D exchange in insulin molecules in the act of docking and integrating with the seeds, proving that the profound structural disruption is the sine qua non of forming an aggregation-competent conformation.     

Hydration from hydrodynamics. General considerations and applications of bead modelling to globular proteins.

The effect of hydration on hydrodynamic properties of globular proteins can be expressed in terms of two quantities: the delta (g/g) parameter and the thickness of the hydration layer. The two paradigms on hydration that originate these alternative measures are described and compared. For the numerical calculation of hydrodynamic properties, from which estimates of hydration can be made, we employ the bead modelling with atomic resolution implemented in programs HYDROPRO and HYDRONMR. As typical, average values, we find 0.3 g/g and a thickness of only approximately 1.2 A. However, noticeable differences in this parameter are found from one protein to another. We have made a numerical analysis, which leaves apart marginal influences of modelling imperfections by simulating properties of a spherical protein. This analysis confirms that the errors that one can attribute to the experimental quantities suffice to explain the observed fluctuations in the hydration parameters. However, for the main purpose of predicting protein solution properties, the above mentioned typical values may be safely used. Particularly for atomic bead modelling, a hydrodynamic radius of approximately 3.2 A yields predictions in very good agreement with experiments.     

Shear-induced self-assembly of native silk proteins into fibrils studied by atomic force microscopy

Noncontact mode atomic force microscopy was used to investigate native silk proteins prepared in different ways. Low protein concentrations revealed that single protein molecules exhibit a simple, round shape with apparent diameters of 20-25 nm. Shearing the native protein solutions after extraction from the gland and prior to drying led to a beads-on-a-string assembly at the nanometer scale. Protein concentration had a significant effect on the morphology of the protein assemblies. At higher protein concentrations, shear-induced alignment into nanofibrils was observed, while lower concentrations lead to the formation of much thinner fibrils with a width of about 8 nm.     

Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain

The shape of enveloped viruses depends critically on an internal protein matrix, yet it remains unclear how the matrix proteins control the geometry of the envelope membrane. We found that matrix proteins purified from Newcastle disease virus adsorb on a phospholipid bilayer and condense into fluidlike domains that cause membrane deformation and budding of spherical vesicles, as seen by fluorescent and electron microscopy. Measurements of the electrical admittance of the membrane resolved the gradual growth and rapid closure of a bud followed by its separation to form a free vesicle. The vesicle size distribution, confined by intrinsic curvature of budding domains, but broadened by their merger, matched the virus size distribution. Thus, matrix proteins implement domain-driven mechanism of budding, which suffices to control the shape of these proteolipid vesicles.     

Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion

In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state.     

The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component proteins

Rice dwarf virus (RDV), the causal agent of rice dwarf disease, is a member of the genus Phytoreovirus in the family Reoviridae. RDV is a double-shelled virus with a molecular mass of approximately 70 million Dalton. This virus is widely prevalent and is one of the viruses that cause the most economic damage in many Asian countries. The atomic structure of RDV was determined at 3.5 A resolution by X-ray crystallography. The double-shelled structure consists of two different proteins, the core protein P3 and the outer shell protein P8. The atomic structure shows structural and electrostatic complementarities between both homologous (P3-P3 and P8-P8) and heterologous (P3-P8) interactions, as well as overall conformational changes found in P3-P3 dimer caused by the insertion of amino-terminal loop regions of one of the P3 protein into the other. These interactions suggest how the 900 protein components are built into a higher-ordered virus core structure.     

Selenocysteine biosynthesis and mechanism of incorporation into growing proteins

The universal genetic code codes for the 20 canonical amino acids, while selenocysteine (Sec) is encoded by UGA, one of the three well-known stop codons. Selenocysteine is of particular interest of molecular biology, principally differing in the mechanism of incorporation into growing polypeptide chains from the other 20 amino acids. The process involves certain cis- and trans-active factors, such as the Sec insertion sequence (SECIS). The SECIS is in the 3′-untranslated mRNA region in eukaryotes and within the open reading frame located immediately downstream of the Sec UGA codon in bacteria, the difference leading to differences in the mechanism of Sec incorporation between the two domains of life. The trans-active factors include Sec-tRNA^[Ser]Sec, which is synthesized by a unique system; the Sec-specific elongation factor EFsec; and a SECIS-binding protein (SBP2). Thus, many additional molecules are to be synthesized in the cell to allow Sec incorporation during translation. The fact makes Sec-containing proteins rather “expensive” and emphasizes their crucial role in metabolism.