Amyloid formation may involve alpha- to beta sheet interconversion via peptide plane flipping

The toxic component of amyloid is not the mature fiber but a soluble prefibrillar intermediate. It has been proposed, from molecular dynamics simulations, that the precursor is composed of alpha sheet, which converts into the beta sheet of mature amyloid via peptide plane flipping. alpha sheet, not seen in proteins, occurs as isolated stretches of polypeptide. We show that the alpha- to beta sheet transition can occur by the flipping of alternate peptide planes. The flip can be described as alphaRalphaL<-->betabeta. A search conducted within sets of closely related protein crystal structures revealed that these flips are common, occurring in 8.5% of protein families. The average "alphaL" conformation found is in an adjacent and less populated region of the Ramachandran plot, as expected if the flanking peptide planes, being hydrogen bonded, are restricted in their movements. This work provides evidence for flips allowing direct alpha- to beta sheet interconversion.     

Designing supramolecular protein assemblies

Many natural proteins self-assemble, either to fulfill their biological function or as part of a pathogenic process. Biological assembly phenomena such as amyloidogenesis, domain swapping and symmetric oligomerization are inspiring new strategies for designing proteins that self-assemble to form supramolecular complexes. Recent advances include the design of novel proteins that assemble into filaments, symmetric cages and regular arrays.     

Conformational dynamics of supramolecular protein assemblies

Supramolecular protein assemblies including molecular motors, cytoskeletal filaments, chaperones, and ribosomes play a central role in a broad array of cellular functions ranging from cell division and motility to RNA and protein synthesis and folding. Single-particle reconstructions of such assemblies have been growing rapidly in recent years, providing increasingly high resolution structural information under native conditions. While the static structure of these assemblies provides essential insight into their mechanism of biological function, their dynamical motions provide additional important information that cannot be inferred from structure alone. Here we present an unsupervised computational framework for the analysis of high molecular weight protein assemblies and use it to analyze the conformational dynamics of structures deposited in the Electron Microscopy Data Bank. Protein assemblies are modeled using a recently introduced coarse-grained modeling framework based on the finite element method, which is used to compute equilibrium thermal fluctuations, elastic strain energy distributions associated with specific conformational transitions, and dynamical correlations in distant molecular domains. Results are presented in detail for the ribosome-bound termination factor RF2 from Escherichia coli, the nuclear pore complex from Dictyostelium discoideum, and the chaperonin GroEL from E. coli. Elastic strain energy distributions reveal hinge-regions associated with specific conformational change pathways, and correlations in collective molecular motions reveal dynamical coupling between distant molecular domains that suggest new, as well as confirm existing, allosteric mechanisms. Results are publically available for use in further investigation and interpretation of biological function including cooperative transitions, allosteric communication, and molecular mechanics, as well as in further classification and refinement of electron microscopy based structures.     

Molecular basis of RADAR anti-phage supramolecular assemblies

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Inhibitory effect of supramolecular polyrotaxane-dipeptide conjugates on digested peptide uptake via intestinal human peptide transporter

The effect of polyrotaxane-dipeptide (Val-Lys) conjugates on the uptake of a model dipeptide (Gly-Sar) was examined via human peptide transporter (hPEPT1) on HeLa cells. Here, Val-Lys groups are introduced to alpha-CDs, which are threaded onto a poly(ethylene oxide) chain capped with bulky end-groups (polyrotaxane). The Gly-Sar uptake via hPEPT1 was significantly inhibited in the polyrotaxane conjugates, and this inhibitory effect was not explained by the sum of interaction between hPEPT1 and alpha-CD-Val-Lys conjugates. Further, the inhibition was significantly greater than those observed in dextran-Val-Lys conjugates. Therefore, our data clearly suggests that supramolecular structure in the polyrotaxane conjugates contributes considerably to the inhibitory effect via multivalent binding of Val-Lys groups with hPEPT1.     

Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein beta-2-microglobulin at pH 7.0 and 37 degrees C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans-proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect beta-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.     

Visualization and classification of amyloid beta supramolecular assemblies

Deposition of amyloid beta (Abeta) fibrils has been suggested to play a central role in Alzheimer's disease. In clarifying the mechanism by which fibrils form and moreover in developing new treatments for amyloidosis, direct observation is important. Focusing on the interactions with surfaces at the early stages, we studied the spontaneous formation of Abeta(1-40) fibrils on quartz slides, monitored by total internal reflection fluorescence microscopy combined with thioflavin T, an amyloid-specific fluorescence dye. Self-assembly of Abeta(1-40), accelerated by a low concentration of sodium dodecyl sulfate, produced various remarkable amyloid assemblies. Densely packed spherulitic structures with radial fibril growth were typically observed. When the packing of fibrils was coarse, extremely long fibrils often protruded from the spherulitic cores. In other cases, a large number of wormlike fibrils were formed. Transmission electron microscopy and atomic force microscopy revealed relatively short and straight fibrillar blocks associated laterally without tight interaction, leading to random-walk-like fibril growth. These results suggest that, during spontaneous fibrillation, the nucleation occurring in contact with surfaces is easily affected by environmental factors, creating various types of nuclei, and hence variations in amyloid morphology. A taxonomy of amyloid supramolecular assemblies will be useful in clarifying the structure-function relationship of amyloid fibrils.     

Access to hybrid supramolecular salen-porphyrin assemblies via a selective in situ transmetalation-metalation self-assembly sequence

New supramolecular hybrid assemblies have been selectively prepared starting off with stoichiometric mixtures of pyridine-tagged Zn(salphen) and non-metalated porphyrin ligands in the presence of suitable metal acetates. This one-pot procedure leads to in situ transmetalation of the salen unit, metalation of the porphyrin ligand by the released Zn(OAc)₂ and subsequent heterometallic assembly formation between the metallosalen and metalloporphyrin components via Zn-N_pyr coordination motifs. The reactions were followed by NMR spectroscopy and MALDI-TOF mass spectrometry, and for one of the Zn(salphen) building blocks the X-ray molecular structure is reported.     

Emergent functions of proteins in non-stoichiometric supramolecular assemblies

Proteins are the basic functional units of the cell, carrying out myriads of functions essential for life. There are countless reports in molecular cell biology addressing the functioning of proteins under physiological and pathological conditions, aiming to understand life at the atomistic-molecular level and thereby being able to develop remedies against diseases. The central theme in most of these studies is that the functional unit under study is the protein itself. Recent rapid progress has radically challenged and extended this protein-function paradigm, by demonstrating that novel function(s) may emerge when proteins form dynamic and non-stoichiometric supramolecular assemblies. There is an increasing number of cases for such collective functions, such as targeting, localization, protection/shielding and filtering effects, as exemplified by signaling complexes and prions, biominerals and mucus, amphibian adhesions and bacterial biofilms, and a broad range of membraneless organelles (bio-condensates) formed by liquid-liquid phase separation in the cell. In this short review, we show that such non-stoichiometric organization may derive from the heterogeneity of the system, a mismatch in valency and/or geometry of the partners, and/or intrinsic structural disorder and multivalency of the component proteins. Either way, the resulting functional features cannot be simply described by, or predicted from, the properties of the isolated single protein(s), as they belong to the collection of proteins.     

Exploiting genomic patterns to discover new supramolecular protein assemblies

Bacterial microcompartments are supramolecular protein assemblies that function as bacterial organelles by compartmentalizing particular enzymes and metabolic intermediates. The outer shells of these microcompartments are assembled from multiple paralogous structural proteins. Because the paralogs are required to assemble together, their genes are often transcribed together from the same operon, giving rise to a distinctive genomic pattern: multiple, typically small, paralogous proteins encoded in close proximity on the bacterial chromosome. To investigate the generality of this pattern in supramolecular assemblies, we employed a comparative genomics approach to search for protein families that show the same kind of genomic pattern as that exhibited by bacterial microcompartments. The results indicate that a variety of large supramolecular assemblies fit the pattern, including bacterial gas vesicles, bacterial pili, and small heat‐shock protein complexes. The search also retrieved several widely distributed protein families of presently unknown function. The proteins from one of these families were characterized experimentally and found to show a behavior indicative of supramolecular assembly. We conclude that cotranscribed paralogs are a common feature of diverse supramolecular assemblies, and a useful genomic signature for discovering new kinds of large protein assemblies from genomic data.     

Chiroptical property tuning of supramolecular assemblies in polymer matrices

Chiroptical materials have received much attention in diverse fields for applications such as displays, sensors, smart memory devices, and catalysis. Here, we develop a simple fabrication method for polymer films with tunable chiroptical properties using small amounts of self-assembling fluorescent dye as an additive. Both the circular dichroism and circularly polarized luminescence signals of the film can be tuned between positive and negative values by thermal treatment. The chiroptical properties can be varied by slight changes in the orientation of chiral pyrene moieties in self-assembled nanofibril networks.     

Amyloid fibril formation by peptide LYS (11-36) in aqueous trifluoroethanol

Peptide LYS (11-36), derived from the beta-sheet region of T4 lysozyme, forms an amyloid fibril in aqueous trifluoroethanol (TFE) at elevated temperature. The peptide has a moderate alpha-helix content in 20 and 50% (v/v) TFE solution; large quantities of fibrils were formed after incubation at 55 degrees C for 2 weeks as monitored by a thioflavin T fluorescence assay. No fibrils were observed when the peptide initially existed predominantly as a random coil or as a complete alpha helix. Our results suggest that a moderate amount of alpha helix and random coil present in the peptide initially facilitates the fibril-formation process, but a high alpha-helix content inhibits fibril formation. Transmission electron microscopy revealed several types of fibril morphologies at different TFE concentrations. The fibrils were highly twisted and consisted of interleaved protofilaments in 50% TFE, while smooth and flat ribbonlike fibrils were found in 20% TFE. In 50% TFE, the fibril growth rate of LYS (11-36) was found to depend strongly on peptide concentration and seeding but was insensitive to solution pH and ionic strength.     

Amyloid fibrils compared to peptide nanotubes

Prefibrillar oligomeric states and amyloid fibrils of amyloid-forming proteins qualify as nanoparticles. We aim to predict what biophysical and biochemical properties they could share in common with better researched peptide nanotubes. We first describe what is known of amyloid fibrils and prefibrillar aggregates (oligomers and protofibrils): their structure, mechanisms of formation and putative mechanism of cytotoxicity. In distinction from other neuronal fibrillar constituents, amyloid fibrils are believed to cause pathology, however, some can also be functional. Second, we give a review of known biophysical properties of peptide nanotubes. Finally, we compare properties of these two macromolecular states side by side and discuss which measurements that have already been done with peptide nanotubes could be done with amyloid fibrils as well.     

Formation of framework nacre polypeptide supramolecular assemblies that nucleate polymorphs

The formation of aragonite in the mollusk shell nacre layer is linked to the assembly of framework protein complexes that interact with β-chitin polysaccharide. What is not yet understood is how framework nacre proteins control crystal growth. Recently, a 30 AA intrinsically disordered nacre protein sequence (n16N) derived from the n16 framework nacre protein was found to form aragonite, vaterite, or ACC deposits when adsorbed onto β-chitin. Our present study now establishes that n16N assembles to form amorphous nonmineralized supramolecular complexes that nucleate calcium carbonate polymorphs in vitro. These complexes contain unfolded or disordered (54% random coil, 46% β structures) n16N polypeptide chains that self-assemble in response to alkaline pH shift. The pH-dependent assembly process involves two stages, and it is likely that side chain salt-bridging interactions are a major driving force in n16N self-association. Intriguingly, Ca(II) ions are not required for n16N assembly but do shift the assembly process to higher pH values, and it is likely that Ca(II) plays some role in stabilizing the monomeric form of n16N. Using preassembled fibril-spheroid n16N assemblies on Si wafers or polystyrene supports, we were able to preferentially nucleate vaterite at higher incidence compared to control scenarios, and it is clear that the n16N assemblies are in contact with the nucleating crystals. We conclude that the framework nacre protein sequence n16N assembles to form supramolecular complexes whose surfaces act as nucleation sites for crystal growth. This may represent a general mineralization mechanism employed by framework nacre proteins in general.     

From high-resolution AFM topographs to atomic models of supramolecular assemblies

Atomic force microscopy (AFM) has developed into a powerful tool in membrane biology. AFM features an outstanding signal-to-noise ratio that allows substructures on individual macromolecules to be visualized. Most recently, AFM topographs have shown the supramolecular assembly of the bacterial photosynthetic complexes in native membranes. Here, we have determined the translational and rotational degrees of freedom of the complexes in AFM images of multi-protein assemblies, in order to build realistic atomic models of supramolecular assemblies by docking high-resolution structures into the topographs. Membrane protein assemblies of megadalton size comprising several hundreds of polypeptide chains and pigments were built with Angstrom precision.     

Amyloid formation by salmon calcitonin

It is demonstrated using three independent methods that salmon calcitonin can form amyloid fibrils in vitro. Large aggregates are shown to exhibit a blue-green birefringence in cross polarised light after staining with congo red. Individual fibrils were observed using electron microscopy. These fibrils are approx. 50–60 Å in diameter and up to 20 000 Å in length and are similar in appearance to those observed in Alzheimer's disease. Finally, X-ray diffraction studies of the large aggregates reveal the cross-β conformation characteristic of the monomers in the fibre.     

The formation of cell assemblies

A simple model is presented for the formation of functional groups in a random neural net. They show the following characteristics: 1. They can maintain autonomous activity which might serve as temporary memory traces. 2. Early in the process of formation they become resistant to contraction. 3. Later they become resistant to expansion. 4. Nearby groups inhibit one another. 5. Two groups may contain some cells in common.     

Two coordinatively linked supramolecular assemblies constructed from highly electron deficient porphyrins

The synthesis of a pair of electron-deficient porphyrin building blocks and their resulting coordinatively linked supramolecular assemblies are described. The perfluorophenyl substituted porphyrins feature pendant pyridines which, upon reaction with Re(CO)₅Cl assemble into discrete dimers and tetramers, as dictated by the geometry of the porphyrin monomer. The resulting supramolecular complexes as well as their constituent porphyrins display several interesting and potentially useful properties owing to the electron-withdrawing nature of the perfluorophenyl funtionalities. Structural, spectroscopic, and electrochemical data indicate that the electron-deficient porphyrins remain planar, allowing for modulation of spectral and redox properties, as well as for enhancement of the affinity of the porphyrins for Lewis-basic ligands.