AFM Probing of Amyloid-Beta 42 Dimers and Trimers

Elucidating the molecular mechanisms in the development of such a devastating neurodegenerative disorder as Alzheimer\u27s disease (AD) is currently one of the major challenges of molecular medicine. Evidence strongly suggests that the development of AD is due to the accumulation of amyloid β (Aβ) oligomers; therefore, understanding the molecular mechanisms defining the conversion of physiologically important monomers of Aβ proteins into neurotoxic oligomeric species is the key for the development of treatments and preventions of AD. However, these oligomers are unstable and unavailable for structural, physical, and chemical studies. We have recently developed a novel flexible nano array (FNA)-oligomer scaffold approach in which monomers tethered inside a flexible template can assemble spontaneously into oligomers with sizes defined by the number of tethered monomers. The FNA approach was tested on short decamer Aβ(14-23) peptides which were assembled into dimers and trimers. In this paper, we have extended our FNA technique for assembling full-length Aβ42 dimers. The FNA scaffold enabling the self-assembly of Aβ42 dimers from tethered monomeric species has been designed and the assembly of the dimers has been validated by AFM force spectroscopy experiments. Two major parameters of the force spectroscopy probing, the rupture forces and the rupture profiles, were obtained to prove the assembly of Aβ42 dimers. In addition, the FNA-Aβ42 dimers were used to probe Aβ42 trimers in the force spectroscopy experiments with the use of AFM tips functionalized with FNA-Aβ42 dimers and the surface with immobilized Aβ42 monomers. We found that the binding force for the Aβ42 trimer is higher than the dimer (75 ± 7 pN vs. 60 ± 3 pN) and the rupture pattern corresponds to a cooperative dissociation of the trimer. The rupture profiles for the dissociation of the Aβ42 dimers and trimers are proposed. Prospects for further extension of the FNA-based approach for probing of higher order oligomers of Aβ42 proteins are discussed

The Sequence Dependent Nanoscale Structure of CENP-A Nucleosomes

CENP-A is a histone variant found in high abundance at the centromere in humans. At the centromere, this histone variant replaces the histone H3 found throughout the bulk chromatin. Additionally, the centromere comprises tandem repeats of α-satellite DNA, which CENP-A nucleosomes assemble upon. However, the effect of the DNA sequence on the nucleosome assembly and centromere formation remains poorly understood. Here, we investigated the structure of nucleosomes assembled with the CENP-A variant using Atomic Force Microscopy. We assembled both CENP-A nucleosomes and H3 nucleosomes on a DNA substrate containing an α-satellite motif and characterized their positioning and wrapping efficiency. We also studied CENP-A nucleosomes on the 601-positioning motif and non-specific DNA to compare their relative positioning and stability. CENP-A nucleosomes assembled on α-satellite DNA did not show any positional preference along the substrate, which is similar to both H3 nucleosomes and CENP-A nucleosomes on non-specific DNA. The range of nucleosome wrapping efficiency was narrower on α-satellite DNA compared with non-specific DNA, suggesting a more stable complex. These findings indicate that DNA sequence and histone composition may be two of many factors required for accurate centromere assembly

Site-Search Process for Synaptic Protein-DNA Complexes

The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems

Nano-assembly of amyloid β peptide: role of the hairpin fold.

Structural investigations have revealed that β hairpin structures are common features in amyloid fibrils, suggesting that these motifs play an important role in amyloid assembly. To test this hypothesis, we characterized the effect of the hairpin fold on the aggregation process using a model β hairpin structure, consisting of two Aβ(14-23) monomers connected by a turn forming YNGK peptide. AFM studies of the assembled aggregates revealed that the hairpin forms spherical structures whereas linear Aβ(14-23) monomers form fibrils. Additionally, an equimolar mixture of the monomer and the hairpin assembles into non-fibrillar aggregates, demonstrating that the hairpin fold dramatically changes the morphology of assembled amyloid aggregates. To understand the molecular mechanism underlying the role of the hairpin fold on amyloid assembly, we performed single-molecule probing experiments to measure interactions between hairpin and monomer and two hairpin complexes. The studies reveal that the stability of hairpin-monomer complexes is much higher than hairpin-hairpin complexes. Molecular dynamics simulations revealed a novel intercalated complex for the hairpin and monomer and Monte Carlo modeling further demonstrated that such nano-assemblies have elevated stability compared with stability of the dimer formed by Aβ(14-23) hairpin. The role of such folding on the amyloid assembly is also discussed

Free Cholesterol Accelerates Aβ Self-Assembly on Membranes at Physiological Concentration

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer\u27s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process

Insight Into the Dynamics of APOBEC3G Protein in Complexes with DNA Assessed by High Speed AFM

APOBEC3G (A3G) is a single-stranded DNA (ssDNA) binding protein that restricts the HIV virus by deamination of dC to dU during reverse transcription of the viral genome. A3G has two zinc-binding domains: the N-terminal domain (NTD), which efficiently binds ssDNA, and the C-terminal catalytic domain (CTD), which supports deaminase activity of A3G. Until now, structural information on A3G has been lacking, preventing elucidation of the molecular mechanisms underlying its interaction with ssDNA and deaminase activity. We have recently built a computational model for the full-length A3G monomer and validated its structure using data obtained by time-lapse High-Speed Atomic Force Microscopy (HS AFM). Here time-lapse HS AFM is applied to directly visualize the structure and dynamics of A3G in complexes with ssDNA. Our results demonstrate a highly dynamic structure of A3G, where two domains of the protein fluctuate between compact globular and extended dumbbell structures. Quantitative analysis of our data revealed a substantial increase in the number of A3G dumbbell structures in the presence of the DNA substrate, suggesting that the interaction of A3G with the ssDNA substrate stabilizes this dumbbell structure. Based on these data, we proposed a model explaining the interaction of globular and dumbbell structures of A3G with ssDNA and suggested a possible role of the dumbbell structure in A3G function

Effect of Histone H4 Tail on Nucleosome Stability and Internucleosomal Interactions

Chromatin structure is dictated by nucleosome assembly and internucleosomal interactions. The tight wrapping of nucleosomes inhibits gene expression, but modifications to histone tails modulate chromatin structure, allowing for proper genetic function. The histone H4 tail is thought to play a large role in regulating chromatin structure. Here we investigated the structure of nucleosomes assembled with a tail-truncated H4 histone using Atomic Force Microscopy. We assembled tail-truncated H4 nucleosomes on DNA templates allowing for the assembly of mononucleosomes or dinucleosomes. Mononucleosomes assembled on nonspecific DNA led to decreased DNA wrapping efficiency. This effect is less pronounced for nucleosomes assembled on positioning motifs. Dinucleosome studies resulted in the discovery of two effects- truncation of the H4 tail does not diminish the preferential positioning observed in full-length nucleosomes, and internucleosomal interaction eliminates the DNA unwrapping effect. These findings provide insight on the role of histone H4 in chromatin structure and stability

Site-specific labeling of supercoiled DNA

Visualization of site-specific labels in long linear or circular DNA allows unambiguous identification of various local DNA structures. Here we describe a novel and efficient approach to site-specific DNA labeling. The restriction enzyme SfiI binds to DNA but leaves it intact in the presence of calcium and therefore may serve as a protein label of 13 bp recognition sites. Since SfiI requires simultaneous interaction with two DNA recognition sites for stable binding, this requirement is satisfied by providing an isolated recognition site in the DNA target and an additional short DNA duplex also containing the recognition site. The SfiI/DNA complexes were visualized with AFM and the specificity of the labeling was confirmed by the length measurements. Using this approach, two sites in plasmid DNA were labeled in the presence of a large excess of the helper duplex to compete with the formation of looped structures of the intramolecular synaptic complex. We show that the labeling procedure does not interfere with the superhelical tension-driven formation of alternative DNA structures such as cruciforms. The complex is relatively stable at low and high pH (pH 5 and 9) making the developed approach attractive for use at conditions requiring the pH change