Mesoscale Ordering of Phycocyanin Molecules in Langmuir-Blodgett Multilayers.

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir-Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir-Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir-Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder. Scanning X-ray nanodiffraction suggests that structural transformation is not homogeneous through the film but limited to patches of up to about 10 µm diameter.

Langmuir-Blodgett nanotemplates for protein crystallography.

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins). We describe how to produce highly ordered 2D LB protein monolayers at the air-water interface and deposit them on glass slides. LB-film formation can be observed by surface-pressure measurements and Brewster angle microscopy (BAM), although its quality can be characterized by atomic force microscopy (AFM) and nanogravimetry. Such films are then used as a 2D template for triggering 3D protein crystal formation by hanging-drop vapor diffusion. The procedure for forming the 2D template takes a few minutes. Structural information about the protein reorganization in the LB film during the crystallization process on the nano level can be obtained using an in situ submicron GISAXS (grazing-incidence small-angle X-ray scattering) method. MicroGISAXS spectra, measured directly at the interface of the LB films and protein solution in real time, as described in this protocol, can be interpreted in terms of the buildup of layers, islands, or holes. In our experience, the obtained LB crystals take 1-10 d to prepare and they are more ordered and radiation stable as compared with those produced using other crystallization methods.

Langmuir-Blodgett (LB)-based nanobiocrystallography at the frontiers of cancer proteomics.

BACKGROUND/AIM: Langmuir-Blodgett (LB) films used as templates for crystallization lead to marked changes in protein stability and water dehydration, despite slight changes in protein atomic structure. Herein, we discuss the importance of LB-based nanocrystallography at the frontiers of cancer proteomics focusing on two model proteins with important biological roles in cancer, namely CK2alpha and RNase A. MATERIALS AND METHODS: Computational mutagenesis using the KINARI Mutagen webserver exhibits different behaviors in terms of stability and robustness, as well as in terms of water dynamics. CONCLUSION: Introduction of LB film leads to the appearance of water molecules close to the protein surface with larger volume, causing changes in crystal stability against radiation and appearing replicated in mutant proteins. Implications for drug design, drug delivery and cancer-causing protein variants are herein presented, along with a review of the most recent findings in LB-based nanobiocrystallography.

Identification of best protein crystallization methods by molecular dynamics (MD).

A full-atom structure of a protein provides an important piece of information for molecular biologists, but has to be complemented by further knowledge concerning its conformational mobility and functional properties. Some scholars have proposed to integrate proteomics-derived data (mainly obtained with techniques like X-ray and NMR crystallography) with protein bioinformatics and computational approaches, above all molecular dynamics (MD), in order to gain better elucidations about proteins. MD simulations have been applied to different areas of protein sciences, but so far few efforts have been made to couple MD with an understanding of the different crystallization techniques that have been proposed during the decades, like classical vapor diffusion hanging drop and its variants (such as sitting drop), in space- and LB (Langmuir-Blodgett)-based crystallization procedures. Using MD, we show here that the optimal protein crystallization techniques prove to be significantly those based on the LB nanotemplate and on space when compared to the classical vapour diffusion hanging drop and its variants.

A review of the strategies for obtaining high-quality crystals utilizing nanotechnologies and microgravity.

Crystallization is a highly demanding and time-consuming task that causes a real bottle-neck in basic research. Great effort has been made to understand the factors and parameters that influence this process and to finely tune them to facilitate crystal growth. Different crystallization techniques have been proposed over the past decades, such as the classical vapor hanging drop method, its variant the sitting drop method, dialysis, cryo-temperature, gel, batch, and the innovative microgravity (space) techniques like free interface diffusion (FID) and counter-ion diffusion (CID). Here, we present a review of the strategies utilizing Langmuir-Blodgett (LB)-based nanotechnologies, and microgravity techniques for obtaining optimal high-quality crystals, as proven by molecular dynamics (MD) and bioinformatics approaches, namely using a clustering algorithm and protein alignment.

Proteomics and proteogenomics approaches for oral diseases.

Design and implementation of new biocompatible materials and achievements in the field of nanogenomics and nanoproteomics as well as in other related and allied sciences in the broader framework of translational and clinical nanomedicine are paving new avenues for nanodentistry. Classical dentistry is becoming more predictive, preventive, personalized, and participatory, providing the patients with a tailored and targeted treatment and handling of their diseases. Considering the global impact of the oral pathologies, being particularly heavy in underdeveloped and developing countries, it is mandatory from an ethical perspective to ensure a global oral health. Nanobiotechnologies play a major role in this ambitious goal. In this review, we will focus on the bioinformatics, nanogenomics, and nanoproteomics aspects of contemporary nanodentistry, emphasizing the urgent need for an integrated proteogenomics approach and addressing its clinical and translational implications and new future perspectives and scenarios.

Advances in nanocrystallography as a proteomic tool.

In order to overcome the difficulties and hurdles too much often encountered in crystallizing a protein with the conventional techniques, our group has introduced the innovative Langmuir-Blodgett (LB)-based crystallization, as a major advance in the field of both structural and functional proteomics, thus pioneering the emerging field of the so-called nanocrystallography or nanobiocrystallography. This approach uniquely combines protein crystallography and nanotechnologies within an integrated, coherent framework that allows one to obtain highly stable protein crystals and to fully characterize them at a nano- and subnanoscale. A variety of experimental techniques and theoretical/semi-theoretical approaches, ranging from atomic force microscopy, circular dichroism, Raman spectroscopy and other spectroscopic methods, microbeam grazing-incidence small-angle X-ray scattering to in silico simulations, bioinformatics, and molecular dynamics, has been exploited in order to study the LB-films and to investigate the kinetics and the main features of LB-grown crystals. When compared to classical hanging-drop crystallization, LB technique appears strikingly superior and yields results comparable with crystallization in microgravity environments. Therefore, the achievement of LB-based crystallography can have a tremendous impact in the field of industrial and clinical/therapeutic applications, opening new perspectives for personalized medicine. These implications are envisaged and discussed in the present contribution.

Conductometric monitoring of protein-protein interactions.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.

Atomic force microscopy and anodic porous allumina of nucleic acid programmable protein arrays.

The methodological aspects are here presented for the NAPPA (Nucleic Acid Programmable Protein Arrays) characterization by atomic force microscopy and anodic porous alumina. Anodic Porous Alumina represents also an advanced on chip laboratory for gene expression contained in an engineered plasmid vector. The results obtained with CdK2, CDKN1A, p53 and Jun test genes expressed on NAPPA and the future developments are discussed in terms of our pertinent and recent Patents and of their possibility to overcome some limitations of present fluorescence detection in probing protein-protein interaction in both basic sciences and clinical studies.

Analysis of gene expression on anodic porous alumina microarrays.

This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays.

Recombinant laccase: I. Enzyme cloning and characterization.

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-ß-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II.

Prototypes of newly conceived inorganic and biological sensors for health and environmental applications.

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.

Recombinant laccase: II. Medical biosensor.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.

Langmuir-Blodgett nanotemplate and radiation resistance in protein crystals: state of the art.

A state-of-the-art review of the role of the Langmuir-Blodgett nanotemplate on protein crystal structures is here presented. Crystals grown by nanostructured template appear more radiation resistant than the classical ones, even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The electron density maps and the changes in parameters such as total diffractive power, B-factor, and pairwise R-factor have been discussed. Protein crystals, grown by the Langmuir-Blodgett nanotemplate-based method, proved to be more radiation resistant compared to crystals grown by the classical hanging drop method in terms of both global and specific damage.

Nanoproteomics enabling personalized nanomedicine.

Nucleic Acid Programmable Protein Arrays utilize a complex mammalian cell free expression system to produce proteins in situ. In alternative to fluorescent-labeled approaches a new label free method, emerging from the combined utilization of three independent and complementary nanotechnological approaches, appears capable to analyze protein function and protein-protein interaction in studies promising for personalized medicine. Quartz Micro Circuit nanogravimetry, based on frequency and dissipation factor, mass spectrometry and anodic porous alumina overcomes indeed the limits of correlated fluorescence detection plagued by the background still present after extensive washes. This could be further optimized by a homogeneous and well defined bacterial cell free expression system capable to realize the ambitious objective to quantify the regulatory protein networks in humans. Implications for personalized medicine of the above label free protein array using different test genes proteins are reported.

Calcium oxide matrices and carbon dioxide sensors.

Homogeneous matrices of calcium oxide (CaO) were prepared by mixing this material with polyethylene glycol (PEG) acting as malleable inert support in order to obtain processable composites. Preliminary tests were carried out to assess the best concentration of CaO in the composite, individuated in the CaO/PEG weight ratio of 1/4. Experimental data highlighted that the composite was able to selectively detect carbon dioxide (CO(2)) via a nanogravimetric method by performing the experiments inside an atmosphere-controlled chamber filled with CO(2). Furthermore, the composite material showed a linear absorption of CO(2) as a function of the gas concentration inside the atmosphere-controlled chamber, thus paving the way for the possible use of these matrices for applications in the field of sensor devices for long-term evaluation of accumulated environmental CO(2).

Unique water distribution of Langmuir-Blodgett versus classical crystals.

Langmuir-Blodgett films when used as nanotemplates for crystallization often leads to marked changes in protein stability and structure. Earlier we found that stability of proteins is also correlated with aqueous surroundings in the crystals. Here we study the direct relationships between presence of LB nanotemplates and unique patterns of water molecules surrounding the protein, for four model proteins for which 3D structures are available, and where crystallization conditions for each protein are the same except the presence of LB nanotemplate. Shape of frequency distribution of volumes occupied by water molecules were analyzed. They were found to be different between "classical" samples of different proteins, but surprisingly quite similar for LB samples. Volumes occupied by each water molecule as the function of the distance of the given molecule from the protein surface were studied. Introduction of LB film leads to appearance of water molecules close to protein surface but occupying large volumes. These findings confirm earlier experimental findings on the role of water molecules in determining protein stability and thereby pointing to water as a possible candidate for differences apparent in LB crystal stability against radiation.

Oxygen-bound Hell's gate globin I by classical versus LB nanotemplate method.

X-ray atomic structure of recombinant Hell's gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X-ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.65 Å, while classical crystals showed only 3.2 Å resolution. Moreover, the reproducibility of this result in the case of LB crystals was much better-nine crystals from 10 gave the same structural results, while only two of 10 classical crystals were appropriate for the X-ray structure resolution.