Measuring the Conformation and Persistence Length of Single-Stranded DNA Using a DNA Origami Structure.

Measuring the mechanical properties of single-stranded DNA (ssDNA) is a challenge that has been addressed by different methods lately. The short persistence length, fragile structure, and the appearance of stem loops complicate the measurement, and this leads to a large variability in the measured values. Here we describe an innovative method based on DNA origami for studying the biophysical properties of ssDNA. By synthesizing a DNA origami structure that consists of two rigid rods with an ssDNA segment between them, we developed a method to characterize the effective persistence length of a random-sequence ssDNA while allowing the formation of stem loops. We imaged the structure with an atomic force microscope (AFM); the rigid rods provide a means for the exact identification of the ssDNA ends. This leads to an accurate determination of the end-to-end distance of each ssDNA segment, and by fitting the measured distribution to the ideal chain polymer model we measured an effective persistence length of 1.98 ± 0.72 nm. This method enables one to measure short or long strands of ssDNA, and it can cope with the formation of stem loops that are often formed along ssDNA. We envision that this method can be used for measuring stem loops for determining the effect of repetitive nucleotide sequences and environmental conditions on the mechanical properties of ssDNA and the effect of interacting proteins with ssDNA. We further noted that the method can be extended to nanoprobes for measuring the interactions of specific DNA sequences, because the DNA origami rods (or similar structures) can hold multiple fluorescent probes that can be easily detected.

Fractal properties of cell surface structures: A view from AFM.

Cell surfaces are densely populated with various proteins. Aggregation of these proteins to nanoscale clusters can be critical for various cellular functions such as signaling, motility and division. Quantitative characterization of corresponding structures and their changes might be useful to understand these basic cell processes and serve as an early marker of cellular stress or diseases. Atomic force microscopy (AFM) allows high-resolution imaging of cell surface structures, resolving fine details of these structures. Moreover, AFM enables simultaneous imaging of cell surface morphology and mapping of its' mechanical characteristics. This review focuses on applying the fractal dimension measure as a sensitive method to quantify single cell surface structures and their changes from AFM images.

Multiparametric AFM reveals turgor-responsive net-like peptidoglycan architecture in live streptococci.

Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.

Zn2+-Aß40 complexes form metastable quasi-spherical oligomers that are cytotoxic to cultured hippocampal neurons.

The roles of metal ions in promoting amyloid ß-protein (Aß) oligomerization associated with Alzheimer disease are increasingly recognized. However, the detailed structures dictating toxicity remain elusive for Aß oligomers stabilized by metal ions. Here, we show that small Zn(2+)-bound Aß1-40 (Zn(2+)-Aß40) oligomers formed in cell culture medium exhibit quasi-spherical structures similar to native amylospheroids isolated recently from Alzheimer disease patients. These quasi-spherical Zn(2+)-Aß40 oligomers irreversibly inhibit spontaneous neuronal activity and cause massive cell death in primary hippocampal neurons. Spectroscopic and x-ray diffraction structural analyses indicate that despite their non-fibrillar morphology, the metastable Zn(2+)-Aß40 oligomers are rich in ß-sheet and cross-ß structures. Thus, Zn(2+) promotes Aß40 neurotoxicity by structural organization mechanisms mediated by coordination chemistry.

Direct visualization of protease action on collagen triple helical structure.

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen (3/4) fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.

Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging.

One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus-HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine-phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

Controlling the anisotropic magnetic dipolar interactions of PbSe self-assembled nanoparticles on GaAs.

We report on the observation of an anisotropic magnetic dipolar interaction that results from binding PbSe nanoparticles (NPs) to GaAs surfaces by an organic linker. The observed dependence of the blocking temperature on the alignment of the linking molecule relative to the surface normal indicates that the anisotropy is caused by the attachment of the organic linker to the NPs. The presented results may serve as a strategy for fine-tuning the magnetic interactions and anisotropy on surfaces.

Conformational stability and membrane interaction of the full-length ectodomain of HIV-1 gp41: implication for mode of action.

Membrane fusion between the human immunodeficiency virus (HIV) and the target cell plasma membrane is correlated with conformational changes in the HIV gp41 glycoprotein, which include an early exposed conformation (prehairpin) and a late low energy six helix bundle (SHB) conformation also termed hairpin. Peptides resembling regions from the exposed prehairpin have been previously studied for their interaction with membranes. Here we report on the expression, purification, SHB stability, and membrane interaction of the full-length ectodomain of the HIV gp41 and its two deletion mutants, all in their SHB-folded state. The interaction of the proteins with zwitterionic and negatively charged membranes was examined by using various biophysical methods including circular dichroism spectroscopy, differential scanning calorimetry, lipid mixing of large unilamellar vesicles, and atomic force microscopy (AFM). All experiments were done in an acidic environment in which the protein remains in its soluble trimeric state. The data reveal that all three proteins fold into a stable coiled-coil core in aqueous solution and retain a stable helical fold with reduced coiled-coil characteristics in a zwitterionic and negatively charged membrane mimetic environment. Furthermore, in contrast with the extended exposed N-terminal domain, the folded gp41 ectodomain does not induce lipid mixing of zwitterionic membranes. However, it disrupts and induces lipid mixing of negatively charged phospholipid membranes (approximately 100-fold more effective than fusion peptide alone), which are known to be expressed more in HIV-1-infected T cells or macrophages. The results support the emerging model in which one of the roles of gp41 folding into the SHB conformation is to slow down membrane disruption effects induced by early exposed gp41. However, it can further affect membrane morphology once exposed to negatively charged membranes during late stages.

Prevention of Staphylococcus aureus biofilm on dialysis catheters and adherence to human cells.

BACKGROUND: Dialysis patients, often carriers of Staphylococcus aureus in their nares, are at high risk of S. aureus infections. METHODS: We examined whether RNAIII inhibiting peptide (RIP), which interferes with quorum sensing mechanisms, reduces adherence of S. aureus to host cells and to dialysis catheter polymers in vitro. Adherence was tested by spectroscopy using safranin staining, by confocal scanning laser microscopy and by atomic force microscopy. RESULTS: RIP inhibited bacterial adherence to HaCat and HEp-2 cells and reduced adherence and biofilm formation not only on polystyrene, but also on both polyurethane- and silicone-made dialysis catheters, with a preponderant effect on silicone, to which bacteria were more adherent. CONCLUSION: RIP opens a new perspective in anti-S. aureus prophylaxis, particularly in dialysis patients.