Alejandro Barrantes

Interaction between Alzheimer's Aβ1-42 Peptide and DNA Detected by Surface Plasmon Resonance

Alzheimers disease is a form of senile mental disorder characterized by the presence of extracellular plaques, containing amyloid-beta (Abeta) as the main component. According to the amyloid hypothesis, an increase of extracellular Abeta production is in the origin of the aberrant plaques causing neuronal loss and dementia. However, a wealth of evidence has been accumulated pointing to the toxicity of soluble intracellular Abeta, having different morphologies of aggregation, as the origin of the neurodegenerative process. The exact nature of the initial molecular events by which Abeta exerts its neurotoxicity, remains obscure. Different forms of soluble Abeta peptide aggregates have been recently found to reside in the nucleus of CHO cells and Alzheimers disease brain samples. This paper focus mainly on the interaction between DNA and the 42 residue Abeta (Abeta42) as studied by Surface Plasmon Resonance. Electronic microscopy and UV-visible spectroscopy are also used to further characterize the interaction. Particular attention is paid to the extent of Abeta42 aggregation needed to observe the interaction with DNA. Our results show that DNA binds all soluble aggregate forms of Abeta42, therefore suggesting that DNA binding is a general property of different soluble forms of Abeta42, unrelated to the extent of aggregation.

Interfacial Behavior and Activity of Laccase and Bilirubin Oxidase on Bare Gold Surfaces

Two blue multicopper oxidases (MCOs) (viz. Trametes hirsuta laccase (ThLc) and Myrothecium verrucaria bilirubin oxidase (MvBOx)) were immobilized on bare polycrystalline gold (Au) surfaces by direct adsorption from both dilute and concentrated enzyme solutions. The adsorption was studied in situ by means of null ellipsometry. Moreover, both enzyme-modified and bare Au electrodes were investigated in detail by atomic force microscopy (AFM) as well as electrochemically. When adsorbed from dilute solutions (0.125 and 0.25 mg mL⁻¹ in the cases of ThLc and MvBOx, respectively), the amounts of enzyme per unit area were determined to be ca. 1.7 and 4.8 pmol cm⁻², whereas the protein film thicknesses were determined to be 29 and 30 Å for ThLc and MvBOx, respectively. A well-pronounced bioelectrocatalytic reduction of molecular oxygen (O₂) was observed on MvBOx/Au biocathodes, whereas this was not the case for ThLc-modified Au electrodes (i.e., adsorbed ThLc was catalytically inactive). The initially observed apparent k(cat)(app) values for adsorbed MvBOx and the enzyme in solution were found to be very close to each other (viz. 54 and 58 s⁻¹, respectively (pH 7.4, 25 °C)). However, after 3 h of operation of MvBOx/Au biocathodes, kcatapp dropped to 23 s⁻¹. On the basis of the experimental results, conformational changes of the enzymes (in all likelihood, their flattening on the Au surface) were suggested to explain the deactivation of MCOs on the bare Au electrodes.

Tau aggregation followed by atomic force microscopy and surface plasmon resonance, and single molecule tau-tau interaction probed by atomic force spectroscopy.

Intracellular neurofibrillary tangles, composed mainly of tau protein, and extracellular plaques, containing mostly amyloid-beta, are the two types of protein aggregates found upon autopsy within the brain of Alzheimers disease patients. Polymers of tau protein can also be found in other neurodegenerative disorders known as tauopathies. Tau is a highly soluble protein, intrinsically devoid of secondary or tertiary structure, as many others proteins particularly prone to form fibrillar aggregations. The mechanism by which this unfolded molecule evolves to the well ordered helical filaments has been amply studied. In fact, it is a very slow process when followed in the absence of aggregation inducers. Herein we describe the use of surface plasmon resonance, atomic force microscopy, and atomic force spectroscopy to detect tau-tau interactions and to follow the process of aggregation in the absence of aggregation inducers. Tau-tau interactions are clearly detected, although a very long period of time is needed to observe filaments formation. Tau oligomers showing a granular appearance, however, are observed immediately as a consequence of this interaction. These granular tau oligomers slowly evolve to larger structures and eventually to filaments having a size smaller than those reported for paired helical filaments purified from Alzheimers disease.

Influence of pH on the build-up of poly-L-lysine/heparin multilayers

The effect of pH on the build-up of polyelectrolyte multilayers, PEMs, composed by poly-L-lysine and heparin onto two different substrates, silica and gold, has been studied by means of ellipsometry and quartz crystal microbalance with dissipation, QCM-D. Ellipsometry results indicate that the dry mass grows exponentially with the number of layers, and that this amount is larger as the pH values are raised. From QCM-D data the viscoelastic properties of the multilayered structure have been obtained. These data reflect that PEMs become more viscoelastic as the pH values are increased for silica substrates, while for gold the highest viscoelastic behavior is obtained at neutral pH and the elastic behavior becomes dominant as the pH is further increased or decreased. By combining these two surface techniques it has been also possible to determine the solvent content in the multilayers and reach a deeper understanding of the internal structure.

Bioelectrocatalytic reduction of oxygen at gold nanoparticles modified with laccase.

To characterise bioelectrocatalytic oxygen reduction at gold nanoparticles (AuNPs) modified with Trametes hirsuta laccase (ThLc) combined electrochemical and quartz crystal microbalance measurements have been used. The electrodes with different degrees of AuNP-monolayer coverage, θ, have been studied. In every case of θ close to theoretically possible 44 ThLc molecules adsorbed at 22nm diameter AuNP. The bioelectrocatalytic current was recalculated down to the current at a single AuNP. Unexpectedly, the current at a single AuNP was higher when θ was higher. The maximum current reached at a single AuNP was 31·10(-18)A which corresponds to the enzyme turnover (kcat) 13s(-1). This rate is lower than the homogeneous ThLc turnover (190s(-1)) suggesting partial denaturation of ThLc upon adsorption or that some ThLc are not in DET contact with the electrode surface.

The influence of nanoparticles on enzymatic bioelectrocatalysis

In nearly all papers concerning enzyme–nanoparticle based bioelectronic devices, it is stated that the presence of nanoparticles on electrode surfaces per se enhances bioelectrocatalysis, although the reasons for that enhancement are often unclear. Here, we report detailed experimental evidence that neither an overpotential of bioelectrocatalysis, nor direct electron transfer and bioelectrocatalytic reaction rates for an adsorbed enzyme depend on the size of nanoparticles within the range of 20–80 nm, i.e. for nanoparticles that are considerably larger than the enzyme molecules.

Friction Force Spectroscopy As a Tool to Study the Strength and Lateral Diffusion of Protein Layers

We present a method to study the strength of layers of biological molecules in liquid medium. The method is based on the Friction Force Spectroscopy operation mode of the Atomic Force Microscope. It works by scratching the sample surface at different applied loads while registering the evolution of the sample topography and of the friction between probe and sample. Results are presented for BSA and β-casein monolayers on hydrophobic surfaces. We show how the simultaneous monitoring of topography and friction allows detecting differences not only between the strength of both types of layers, but also between the lateral diffusion of the proteins within these layers. Specifically, β-casein is shown to form stronger layers than BSA. The yield strengths calculated for both of these systems are in the range 50-70 MPa. Moreover, while no lateral diffusion is observed for BSA, we show that β-casein diffuses along the hydrophobic substrates at a rate higher than the scan velocity of the tip (16 μm s(-1) in our case).

Tau Protein Provides DNA with Thermodynamic and Structural Features which are Similar to those Found in Histone-DNA Complex

Tau protein has been proposed as a trigger of Alzheimers disease once it is hyperphosphorylated. However, the role that native tau forms play inside the neuronal nucleus remains unclear. In this work we present results concerning the interaction of tau protein with double-stranded DNA, single-stranded DNA, and also with a histone-DNA complex. The tau-DNA interaction results in a structure resembling the beads-on-a-string form produced by the binding of histone to DNA. DNA retardation assays show that tau and histone induce similar DNA retardation. A surface plasmon resonance study of tau-DNA interaction also confirms the minor groove of DNA as a binding site for tau, similarly to the histone binding. A residual binding of tau to DNA in the presence of Distamycin A, a minor groove binder, suggests the possibility that additional structural domains on DNA may be involved in tau interaction. Finally, DNA melting experiments show that, according to the Zipper model of helix-coil transition, the binding of tau increases the possibility of opening the DNA double helix in isolated points along the chain, upon increasing temperature. This behavior is analogous to histones and supports the previously reported idea that tau could play a protective role in stress situations. Taken together, these results show a similar behavior of tau and histone concerning DNA binding, suggesting that post-translational modifications on tau might impair the role that, by modulating the DNA function, might be attributable to the DNA-tau interaction.

Specific binding of DNA to aggregated forms of Alzheimer's disease amyloid peptides

Anomalous protein aggregation is closely associated to age-related mental illness. Extraneuronal plaques, mainly composed of aggregated amyloid peptides, are considered as hallmarks of Alzheimers disease. According to the amyloid cascade hypothesis, this disease starts as a consequence of an abnormal processing of the amyloid precursor protein resulting in an excess of amyloid peptides. Nuclear localization of amyloid peptide aggregates together with amyloid-DNA interaction, have been repeatedly reported. In this paper we have used surface plasmon resonance and electron microscopy to study the structure and behavior of different peptides and proteins, including β-lactoglobulin, bovine serum albumin, myoglobin, histone, casein and the amyloid-β peptides related to Alzheimers disease Aβ25-35 and Aβ1-40. The main purpose of this study is to investigate whether proneness to DNA interaction is a general property displayed by aggregated forms of proteins, or it is an interaction specifically related to the aggregated forms of those particular proteins and peptides related to neurodegenerative diseases. Our results reveal that those aggregates formed by amyloid peptides show a particular proneness to interact with DNA. They are the only aggregated structures capable of binding DNA, and show more affinity for DNA than for other polyanions like heparin and polyglutamic acid, therefore strengthening the hypothesis that amyloid peptides may, by means of interaction with nuclear DNA, contribute to the onset of Alzheimers disease.

Deposition Kinetics of Bioinspired Phenolic Coatings on Titanium Surfaces

Polyphenols can form functional coatings on a variety of different materials through auto-oxidative surface polymerization in a manner similar to polydopamine coatings. However, the mechanisms behind the coating deposition are poorly understood. We report the coating deposition kinetics of the polyphenol tannic acid (TA) and the simple phenolic compound pyrogallol (PG) on titanium surfaces. The coating deposition was followed in real time over a period of 24 h using a quartz crystal microbalance with dissipation monitoring (QCM-D). TA coatings revealed a multiphasic layer formation: the deposition of an initial rigid layer was followed by the buildup of an increasingly dissipative layer, before mass adsorption stopped after approximately 5 h of coating time. The PG deposition was biphasic, starting with the adsorption of a nonrigid viscoelastic layer which was followed by layer stiffening upon further mass adsorption. Coating evaluation by ellipsometry and AFM confirmed the deposition kinetics determined by QCM-D and revealed maximum coating thicknesses of approximately 50 and 75 nm for TA and PG, respectively. Chemical characterization of the coatings and polymerized polyphenol particles indicated the involvement of both physical and chemical interactions in the auto-oxidation reactions.