Pseudo-peptide amyloid-ß blocking inhibitors: molecular dynamics and single molecule force spectroscopy study.

By combining MD simulations and AFS experimental technique, we demonstrated a powerful approach for rational design and single molecule testing of novel inhibitor molecules which can block amyloid-amyloid binding - the first step of toxic amyloid oligomer formation. We designed and tested novel pseudo-peptide amyloid-ß (Aß) inhibitors that bind to the Aß peptide and effectively prevent amyloid-amyloid binding. First, molecular dynamics (MD) simulations have provided information on the structures and binding characteristics of the designed pseudo-peptides targeting amyloid fragment Aß (13-23). The binding affinities between the inhibitor and Aß as well as the inhibitor to itself have been estimated using Umbrella Sampling calculations. Atomic Force Spectroscopy (AFS) was used to experimentally test several proposed inhibitors in their ability to block amyloid-amyloid binding - the first step of toxic amyloid oligomer formation. The experimental AFS data are in a good agreement with theoretical MD calculations and demonstrate that three proposed pseudo-peptides bind to amyloid fragment with different affinities and all effectively prevent Aß-Aß binding in similar way. We propose that the designed pseudo-peptides can be used as potential drug candidates to prevent Aß toxicity in Alzheimer's disease.

Molecular dynamics simulations and Kelvin probe force microscopy to study of cholesterol-induced electrostatic nanodomains in complex lipid mixtures.

The molecular arrangement of lipids and proteins within biomembranes and monolayers gives rise to complex film morphologies as well as regions of distinct electrical surface potential, topographical and electrostatic nanoscale domains. To probe these nanodomains in soft matter is a challenging task both experimentally and theoretically. This work addresses the effects of cholesterol, lipid composition, lipid charge, and lipid phase on the monolayer structure and the electrical surface potential distribution. Atomic force microscopy (AFM) was used to resolve topographical nanodomains and Kelvin probe force microscopy (KPFM) to resolve electrical surface potential of these nanodomains in lipid monolayers. Model monolayers composed of dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (DOPG), and cholesterol were studied. It is shown that cholesterol changes nanoscale domain formation, affecting both topography and electrical surface potential. The molecular basis for differences in electrical surface potential was addressed with atomistic molecular dynamics (MD). MD simulations are compared the experimental results, with 100 s of mV difference in electrostatic potential between liquid-disordered bilayer (Ld, less cholesterol and lower chain order) and a liquid-ordered bilayer (Lo, more cholesterol and higher chain order). Importantly, the difference in electrostatic properties between Lo and Ld phases suggests a new mechanism by which membrane composition couples to membrane function.

Amyloid-ß (1-40) restores adhesion properties of pulmonary surfactant, counteracting the effect of cholesterol.

A pulmonary surfactant (PS) is a thin lipid-protein film covering the surface of the lung alveoli at the air/liquid interface. The primary purpose of a PS is to control the surface tension of the air/liquid interface and to reduce the work of breathing. High levels of cholesterol in a PS are associated with life-threatening acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Finding therapeutics to counteract the effect of cholesterol in a PS is a matter of contemporary research. In our earlier work, we showed that the addition of amyloid-ß (1-40) (Aß40), the protein implicated in Alzheimer's disease, can reverse the detrimental effects of cholesterol in surfactants by improving multilayer formation and restoring PS surface active properties. We hypothesized that this phenomenon was due to Aß40 improving adhesion properties of a surfactant. In this work we used atomic force spectroscopy to demonstrate that Aß40 counteracts the adhesive properties of a PS compromised by high levels of cholesterol in a PS and helps to restore the functionality of a PS.

Comparison of three competing dynamic force spectroscopy models to study binding forces of amyloid-ß (1-42).

We performed single molecule dynamic force spectroscopy experiments to study the dimerization of two amyloid-ß (1-42) peptides and compared three different theoretical models used to fit experimental data: Bell-Evans, Dudko-Hummer-Szabo, and Friddle-De Yoreo. Using these models we extracted values of the dissociation rate at zero force, k0, and height and the width of the energy barrier, ΔG and xß. We show the importance of including the effect of the linker molecule. All three models corrected for the linker effect give comparable results for xß and show more discrepancy for k0 and ΔG values, ΔG parameter correlates well between Dudko-Hummer-Szabo and Friddle-De Yoreo models but differs for the Bell-Evans model.

Effect of melatonin and cholesterol on the structure of DOPC and DPPC membranes.

The cell membrane plays an important role in the molecular mechanism of amyloid toxicity associated with Alzheimer's disease. The membrane's chemical composition and the incorporation of small molecules, such as melatonin and cholesterol, can alter its structure and physical properties, thereby affecting its interaction with amyloid peptides. Both melatonin and cholesterol have been recently linked to amyloid toxicity. Melatonin has been shown to have a protective role against amyloid toxicity. However, the underlying molecular mechanism of this protection is still not well understood, and cholesterol's role remains controversial. We used small-angle neutron diffraction (SAND) from oriented lipid multi-layers, small-angle neutron scattering (SANS) from unilamellar vesicles experiments and Molecular Dynamics (MD) simulations to elucidate non-specific interactions of melatonin and cholesterol with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) model membranes. We conclude that melatonin decreases the thickness of both model membranes by disordering the lipid hydrocarbon chains, thus increasing membrane fluidity. This result is in stark contrast to the much accepted ordering effect induced by cholesterol, which causes membranes to thicken.

[Structural organization of DNA complexes with proteins HMGB1 and HMGB1-(A+B)].

The interaction between DNA and the nonhistone proteins HMGB1 and HMGB1-(A+B) has been studied using circular dichroism and scanning force microscopy. The recombinant protein HMGB1-(A+B) has no negatively charged C-terminal domain characteristic for HMGB1. Our earlier suggestion about the structural interaction of tandem HMGB1-domains of the recombinant protein with DNA was confirmed. It was shown that the C-terminal part modulates the interactions of HMGB1-domains with DNA. Without the C-terminal sequence, the HMGB1-(A+B) protein forms DNA-protein complexes with the ordered supramolecular structure.

AFM study of interaction forces in supported planar DPPC bilayers in the presence of general anesthetic halothane.

In spite of numerous investigations, the molecular mechanism of general anesthetics action is still not well understood. It has been shown that the anesthetic potency is related to the ability of an anesthetic to partition into the membrane. We have investigated changes in structure, dynamics and forces of interaction in supported dipalmitoylphosphatidylcholine (DPPC) bilayers in the presence of the general anesthetic halothane. In the present study, we measured the forces of interaction between the probe and the bilayer using an atomic force microscope. The changes in force curves as a function of anesthetic incorporation were analyzed. Force measurements were in good agreement with AFM imaging data, and provided valuable information on bilayer thickness, structural transitions, and halothane-induced changes in electrostatic and adhesive properties.

Examination of surface-bound Ku-DNA complexes in an aqueous environment using MAC mode atomic force microscopy.

In the development of biosensors, it is essential to understand how the signal-transducing element may perturb surface-bound proteins and nucleic acids. The tip of the atomic force microscope is such an element in atomic force microscopy. In this paper, we describe the influence of tip-sample interactions on the measured height of the DNA repair protein, Ku, that has been adsorbed onto a mica surface which was submerged in aqueous solution. We find that the measured height of the Ku molecule depends critically on whether or not it is associated with DNA. Additionally, we observed that the conditions (time and concentration) under which Ku is incubated with DNA, affect the appearance (number and type) of the DNA-Ku complexes observed.

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy.

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation.

The interaction of natural calf thymus DNA with Cr(3+) ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr(3+) ion binding mainly to N(7) (G) and to phosphate groups was demonstrated. Psi-type VCD spectra resembling electronic CD (ECD) spectra, which appear during psi-type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large-scale helicity. Atomic force microscopy confirmed DNA condensation by Cr(3+) ions and the formation of tight DNA particles responsible for the psi-type VCD spectra. Upon increasing the Cr(3+) ion concentration the shape of the condensates changed from loose flower-like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr(3+) ions studied. The secondary structure of DNA remained in a B-form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles.

Supported planar bilayer formation by vesicle fusion: the interaction of phospholipid vesicles with surfaces and the effect of gramicidin on bilayer properties using atomic force microscopy.

We have used magnetic alternating current mode atomic force microscopy (MAC-AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.