Search

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 protein nanofilms can serve as templates for nucleation and growth of protein crystals. This functionality can be enhanced by thermal annealing. While surface ordering of the multilayered nanofilms and an improvement of the correlation between the layers during thermal annealing have been revealed by atomic force microscopy and grazing-incidence small-angle x-ray scattering, information on the structure developing in the bulk of nanofilms is lacking. In this paper, we report on scanning x-ray nanodiffraction experiments of penicillin-G-acylase multilayers deposited on Si3N4 membranes and annealed at 150 °C. While the annealed multilayer has remained mostly featureless, we observe locally globular aggregates and filamentous spherulites based on nanofibrillar subunits with cross-β amyloidic motifs.

Application of Microarrays Technology to organ transplantation in human with the aid of statistical and non-statistical analysis, including interaction-based methods, is reviewed here.

Our research relates to the implementation of Nucleic Acid Programmable Protein Array (NAPPA) analysis by matrix assisted laser desorption/ionization mass spectrometry (MALDI MS). Such analysis enables interrogation of protein-protein interactions in a labelfree manner by desorption and ionization of analytes (e.g., proteins synthesized from the immobilized cDNA and/or protein bounded to these). Mass spectrometry, in fact, has the unique advantage of being able to determine not only the presence but also establish the identity of a given ligand. However, the development of a MALDI MS-compatible protein microarray is complex since existing methods for forming protein microarrays do not transfer readily onto a MALDI target. Here we present a novel method for implementing a procedure to analyze protein contents on NAPPA protein microarrays using MALDI TOF mass spectrometry.

X-ray diffraction patterns of multilayered Langmuir-Schaefer (LS) film of penicillin G acylase (PGA) enzyme were acquired at the ID13 of Synchrotron Radiation at ESRF (Grenoble, France).As previously shown by GISAXS and AFM (Pechkova et al, J. Synchrotron Radiation 2009) the Langmuir- multilayered enzyme film appears quite reproducible for the PGA here utilized to monitor their diffraction and structural properties in comparison of those apparent in the same PGA after crystallization by classical methods. Our study do show indeed that the enzyme structures are becoming quite more organized due to the heating and cooling process, which leads to the establishment of long-range order and unique diffraction properties. The dramatic increase of long-range order in the LB multi-layered enzyme films after heating and cooling, made previously apparent by grazing incidence small angle X-ray scattering using microbeam, is here confirmed by these synchrotron diffraction studies at the ID13 beamline, opening the way to bypass the bottleneck of protein crystallization for protein structure determination which is leaving still unsolved large part of important proteins , like the membrane ones.

Protein X-ray crystallography will remain the most powerful method to obtain the protein 3D atomic structures in foreseeable future. However, the production of the protein crystal as well as it quality (order, intensity of diffraction, radiation stability) remains the major problem. Many important proteins including those of life science interest and pharmaceutical industry impact are difficult to crystallize. The second major problem in protein crystallography is radiation damage of obtaining crystals which can only be partially overcome by existing methods. In the present work we use the protein LB nanotemplate crystallization method – generalized procedure for triggering of crystallization of any given protein, which allows to obtain radiation stable and high quality diffracting crystals for further X-ray analysis by synchrotron radiation. We apply LB nanotemplate method to crystallization of L-asparaginase from Rhodospirillum rubrum. This protein has potential application for combined chemical and enzymatic therapy of malignant blood disorders and therefore for new anticancer drug development. We also compare the diffraction quality of asparagines crystal obtained by classical method and LB nanotemplate and report preliminary X-ray diffraction characterization by synchrotron radiation.

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.

Polycrystalline samples of lysozyme were prepared with and without a Langmuir-Blodgett (LB) thin film template via both the hanging drop method and batch crystallisation. Powder diffraction methods are used to compare these samples and to measure their resistance to radiation damage at room temperature. The X-ray induced amorphisation of the samples was followed as a function of time and it was shown that diffraction does not entirely disappear even at very long exposure times. Two distinct kinetic timescales are evident suggesting that early and late stage processes are quite different. Radiation damage was also shown to be localized in the sample in the region where the beam impinges.

Biological Faculty, Lomonosov Moscow State University (MSU), Leninskie gory 1, Moscow 119234, Russia Department of Physics, University of Osnabruck, Barbarastr 7, Osnabruck 49076, Germany Department of Health Sciences (DISSAL), University of Genoa, Via Antonio Pastore 1, Genoa 16132, Italy Laboratories of Biophysics and Nanotechnologies (LBN), Department of Experimental Medicine (DIMES), University of Genoa, Via Antonio Pastore 3, Genoa 16132, Italy Nanoworld Institute Fondazione EL.B.A.Nicolini (NWI-FEN), Largo Redaelli 7, Pradalunga, Bergamo 24100, Italy

Conductometric monitoring of drug-gene and drug-protein interactions is of fundamental importance in the field of molecular pharmacology. Here, we present our main findings and characterizations of an important antiblastic used in neuro-oncology (Temozolomide), interacting with selected proteins that represent predictive biomarkers of the rate survival of the patients, of the outcome of chemotherapy and resistance to drug itself (namely, BRIP1 and MLH1). We use our previously introduced two genes along with previously described Nucleic Acid Programmable Protein Arrays (NAPPA)-based nanoconductometric sensor. We performed a positive control (Temozolomide plus MLH1 protein), a negative control (Temozolomide plus BRIP1 protein) and a multi-gene experiment (Temozolomide plus BRIP1&MLH1 being co-expressed), showing that we are able to properly perform pharmacoproteomics tasks, discriminating each protein and drug unique conductance curve as well as their interactions, even in the presence of multi-proteins being immobilized. Moreover, in the last part of our paper, we used a multiple regression model in order to predict the behavior of Temozolomide when exposed to BRIP1&MLH1 co-expressed and we showed that we are able to predict the drug-protein interaction profile with a good regression coefficient.

Laser-induced microfragmentation of LB nanotemplate-induced protein crystals in glycerol solution resu lts in distinct, coherently diffracting domains. Only c rystals produced according to the Langmuir-Blodgett (LB) nanotemplate technique reveal in all four prot eins being tested (lysozyme, insulin, thaumatin and ribonuclease) domains highly radiation resistant, w hile the crystals produced by the standard hanging drop crystallization method do not. Actually the very sa me laser exposure causes the disappearance of these “classical” protein crystals during the same time f rame of 40 min needed for the laser cutting in all four proteins being tested. The microdiffraction of micr ocrystals prepered by the combination of LangmuirBlodgett and Laser technologies proves that not onl y the Lysozyme survives the process, as shown recently by nanodifraction, but also all three other model p roteins appear to behave similarly well, namely ins ulin, thaumatin and ribonuclease. The result confirms the emerging of a new biophysical technique uniquely usefull for synchrotron radiation studies based on small protein microcrystals uniquely radiation resi stant when prepered by LB nanotemplate and subsequently fragmented by Laser.