Homogeneous Oligomers Research Articles

The stability and dynamics of the Aβ40/Aβ42 interlaced mixed fibrils

The accumulation of fibrillar amyloid-β (Aβ) aggregates in the brain, predominantly comprising 40- and 42-residue amyloid-β (Aβ40 and Aβ42), is a major pathological hallmark of Alzheimer’s disease (AD). Aβ40 and Aβ42 naturally coexist in the brain under normal physiological conditions, and their interplay is generally considered to be a critical factor in the progression of AD. In addition to forming homogeneous oligomers and fibrils, Aβ40 and Aβ42 are also reported to co-assemble into hetero-oligomers and interlaced mixed fibrils, as evidenced by solid-state nuclear magnetic resonance spectroscopy (NMR), high molecular weight mass spectrometry and cross-seeding experiments. However, the exact molecular mechanisms underlying these processes remain unclear. In this study, we have used a recently resolved structurally uniform 1:1 mixture of Aβ40/Aβ42 interlaced mixed fibril as a prototype to gain insights into the molecular-level interactions between Aβ40 and Aβ42. We employed fully atomistic molecular dynamics simulation and compared the results with a homogeneous U-shaped Aβ40 fibrillar model. Our simulations using two different force fields provide conclusive evidence that the Aβ40/Aβ42 interlaced mixed fibril is energetically more favorable than the homogeneous Aβ40 fibrillar model. Furthermore, we also show that the increase in stability observed in the mixed model stems primarily from the packing interfaces and the stacking interfaces between C-termini. Our simulation results provide valuable mechanistic insights that are not readily accessible in experiment and could have significant implications for both the pathogenesis of AD and the development of current therapeutic strategies. Communicated by Ramaswamy H. Sarma

The Capsid Domain of Arc Changes Its Oligomerization Propensity through Direct Interaction with the NMDA Receptor

The activity-regulated cytoskeleton-associated protein, Arc, is highly expressed in neuronal dendrites and is involved in synaptic scaling and plasticity. Arc exhibits homology to the capsid-forming Gag proteins from retroviruses and can encapsulate its own mRNA and transport it to neighboring neurons. However, the molecular events that lead to the assembly of Arc capsids and how the capsid formation is regulated are not known. Here we show that the capsid domain of Arc may transiently form homogeneous oligomers of similar size as capsids formed byfull-length Arc. We determined a high-resolution structure of the monomeric Arc capsid domain and mapped the initial structural change in the oligomerization process to the N-terminal part of the capsid domain. Peptide ligands from the NMDA receptor subunits inhibit oligomerization, which suggests that Arc's ability to transfer mRNA between cells may be regulated by protein-protein interactions at the synapse.

Amyloid-β(1-42)Oligomer Models Developed using Combined Solid State NMR and Sequence Specific Hydroxyl Radical Footprinting Data

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) are the pathogenic species in Alzheimer's disease (AD). However, detailed structural information on these species remains scarce due to low levels of endogenous Aβ oligomers and uncertainties surrounding current in vitro model systems. Herein, we describe a hydroxyl radical footprinting (HRF) study of Aβ42 monomers, dimers, and specially prepared stable and homogeneous oligomers. Specific side chain solvent accessibilities of individual residues in the folded and aggregated forms of Aβ42 are measured with respect to the same residues of Aβ42 in a fully exposed reference state. These data provide residue specific side chain solvent accessibility protection factors and are used in complement with biophysical characterizations and ss-NMR analyses of the same systems. Results are discussed in the context of proposed NMR models of Aβ oligomers with implications towards further development of therapeutic and diagnostic strategies.

Integration of multiple chromophores with native photosynthetic antennas to enhance solar energy capture and delivery

Native length bacterial light-harvesting peptides carrying covalently attached designer chromophores have been created that self-assemble with native bacteriochlorophyll a (BChl a) to afford stable antennas with enhanced spectral coverage. Native (or native-like) α- and β-peptides interact with each other and BChl a to form a heterodimeric (αβ-dyad) unit that can then oligomerize to form biohybrid analogs of the bacterial core light-harvesting complex (LH1). Pairs of distinct synthetic chromophores were incorporated in αβ-dyads at selected distances from the BChl a target site (position 0). Two designs were explored. One design used green-yellow absorbing/emitting Oregon Green at the −34 position (toward the N-terminus relative to the BChl a coordination site) of β and orange-red absorbing/emitting Rhodamine Red at the −20 position of α, which combine with BChl a to give homogeneous oligomers. A second design used two different β-peptide conjugates, one with Oregon Green at the −34 position and the second with a near-infrared absorbing/emitting synthetic bacteriochlorin at the −14 position, which combine with α and BChl a to give a heterogeneous mixture of oligomers. The designs afford antennas with ∼45 to ∼60 pigments, provide enhanced spectral coverage across the visible and near-infrared regions relative to native antennas, and accommodate pigments at remote sites that contribute to solar light harvesting via an energy-transfer cascade. The efficiencies of energy-transfer to the BChl a target in the biohybrid antennas are comparable to native antennas, as revealed by static and time-resolved absorption and emission studies. The results show that the biohybrid approach, where designer chromophores are integrated via semisynthesis with native-like scaffolding, constitutes a versatile platform technology for rapid prototyping of antennas for solar energy capture without the laborious synthesis typically required for creating artificial photosynthetic light-harvesting architectures.

Fiber Formation of a Synthetic Peptide Derived from Spider Dragline of Nephila Clavata

Spider dragline is a high performance biopolymer with exceptional mechanical properties. It is 5 times stronger than stainless wire, and high tensile strength and elasticity. The dragline is formed in the major ampullate gland of Nephila clavata and is composed of two major silk proteins, spidroin I and II. Our previous study suggested that the synthetic peptide derived from Nephila clavata forms large-sized fibers. However, the mechanism associated with spinning and the structure of the dragline silk protein remains to be studied in detail.To further investigate the relationship between structure and fiber formation, the fiber forming regions of spidroin were predicted, based on the hydrophobicity of individual amino acid residues. The candidate peptides were chemically synthesized by a solid-phase method, purified by reversed-phase HPLC, and the structures confirmed by MALD-TOF/MS analysis.The conditions for fiber formation for the candidate peptides were screened in a series of aqueous and organic solvents. Large-sized fibers were obtained when an organic solvent was used, under acidic conditions. However, the peptide was not able to form fibers under basic conditions.To obtain structural information on fiber formation, Circular Dichroism measurements of the synthetic spider peptides were performed. The results suggested that the formation of a β-sheet structure is required for fiber formation of the spider peptide.To characterize the peptide fibers, Dynamic laser light-scattering measurements were carried out under several conditions. The results indicated that the synthetic peptide formed a homogeneous oligomer at the initial time and moved to a large-sized oligomer at the later time.In conclusion, the fiber formation of the synthetic spider peptide occurs in organic solvents under acidic conditions and the synthetic peptide forms a homogeneous large-sized oligomer. The results will be discussed in this paper.

Synthesis of a Dimer of β-(1,4)-l-Arabinosyl-(2S,4R)-4-hydroxyproline Inspired by Art v 1, the Major Allergen of Mugwort

Nα-tert-Butoxycarbonyl-L-trans-4-hydroxyproline allyl ester (Boc-Hyp-OAll) was glycosylated with 2,3,5-tri-O-benzyl-L-arabinose p-cresylthioglycoside in 60% yield with 4:1 β:α stereoselectivity. Deprotection of N- and C-terminii independently gave a prolyl amine and prolyl carboxylate respectively that were coupled under standard conditions with 1-[bis-(dimethylamino)methylene]-1H-1,2,3-triazolo-[4,5,b]-pyridininium hexafluorophosphate 3-oxide (N-HATU) to give the dimer 1 in 46% yield. These results represent the first steps toward the production of homogeneous oligomers to determine the minimal epitope of the Art v 1 allergen.

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Effect of the number of monomers in the chain of an oligomer on the function of its complex formation with polydentate ligands in solutions

Simulated curves of pH-metric and spectrometric titration of homogeneous oligomers were analyzed to reveal the possibility of recognition of distinctions in the distribution diagrams of polymer and oligomer forms. Analysis of experimental data gave grounds to assume that the features of the titration curves of polyadenylic-polyuridylic ribonucleic acid are caused by cleavage of polymeric chains and formation of fairly short oligomers.

Bacillus subtilis division protein DivIVA - screen for stable oligomer state conditions.

The cell division protein DivIVA is predicted to be a coiled-coil, tropomyosin-like protein, that self-associates both in vivo and in vitro into oligomers of up to 10-12 monomers. A simple and quick screen for conditions supporting the stable oligomer structure has been developed revealing that DivIVA forms a homogeneous oligomer in the presence of PEGs (PEG 4 K or PEG 8K and PEG 1K).

Thermodynamic Stability of Human Lens Recombinant αA- and αB-crystallins

Lens alpha-crystallin is a 600-800-kDa heterogeneous oligomer protein consisting of two subunits, alphaA and alphaB. The homogeneous oligomers (alphaA- and alphaB-crystallins) have been prepared by recombinant DNA technology and shown to differ in the following biophysical/biochemical properties: hydrophobicity, chaperone-like activity, subunit exchange rate, and thermal stability. In this study, we studied their thermodynamic stability by unfolding in guanidine hydrochloride. The unfolding was probed by three spectroscopic parameters: absorbance at 235 nm, Trp fluorescence intensity at 320 nm, and far-UV circular dichroism at 223 nm. Global analysis indicated that a three-state model better describes the unfolding behavior than a two-state model, an indication that there are stable intermediates for both alphaA- and alphaB-crystallins. In terms of standard free energy (DeltaG(NU)(H(2)(O))), alphaA-crystallin is slightly more stable than alphaB-crystallin. The significance of the intermediates may be related to the functioning of alpha-crystallins as chaperone-like molecules.

Three-Dimensional Reconstruction of Metal Replicas of theHelicobacter pyloriVacuolating Cytotoxin

TheHelicobacter pylorivacuolating cytotoxin (VacA) forms high molecular weight homooligomers which contain either six or seven copies of a 95-kDa polypeptide. Electron microscope visualization of carbon platinum replicas of quick-freeze, deep-etched, preparations of VacA has revealed that the oligomers are arranged in flower-like structures with six- or sevenfold radial symmetry, depending on the number of 95-kDa oligomers that they contain. Each monomer is structured in two subunits of 37 and 58 kDa connected by an exposed loop which is a site for proteolytic cleavage. In preparations of VacA which had undergone extensive cleavage at the exposed loop, oligomers of both six- and sevenfold symmetry which appeared flatter were observed; these latter were interpreted as molecules which had lost a complete set of one of the subunits. We exploited a 3D reconstruction of metal replicas of quick-freeze, deep-etched, oligomers, representing the four types of molecules described. All the molecules appear to adhere with the same face toward the mica. Images of rotary shadowed oligomers were processed by multivariate statistical analysis to evidence clusters of equivalent and homogeneous oligomers. 3D reconstructions of the replicas so classified were performed by random conical tilt tomography. In the case of intact molecules (not cleaved) the reconstructions represent both the outer and the inner surfaces of the mold; the latter gives a reasonably accurate sense of the upper surface of the VacA oligomers. These data support the hypothesis that VacA is an AB type toxin and suggest a model in which the smaller of the two subunits is arranged in a uniform ring on the surface of the molecule in such a way as to contribute to the overall stability of the molecule.

Aggregation equilibria in solvent extraction of iron(III) halides

Osmometric measurements on benzene solutions containing trilaurylammonium chloride or bromide, TLAH X. and tetrachloro or tetrabromoferrate, TLAHFe X 4, ( X = Cl and Br) and data on the partition of iron(III) chloride between aqueous hydrochloric acid solutions and benzene solutions of trilaurylammonium chloride indicate that in the equilibrium organic phase the predominate metal complexes are TLAHFe X 4 and TLAH X·TLAHFe X 4 in addition to the various homogeneous oligomers of both TLAH X and TLAHFe X 4. Their formation constants are given. The water content of the benzene phase has no significant effect upon the aggregation equilibria.