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Biomedical subjects

Eugenia Pechkova

Publications and source records attributed to Eugenia Pechkova.

13 recordsLinked to original sources
PubMed2006

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: I. microGISAXS and microdiffraction of P450scc.

Ultrasmall P450scc cytochrome microcrystals are grown by classical hanging vapor diffusion and by its modification using homologous protein thin-film template displaying a long-range order. The nucleation and growth mechanisms of P450scc microcrystals are studied at the thin cytochrome film surface by a new microbeam grazing incidence small angle X-ray scattering (microGISAXS) technique developed at the microfocus beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. P450scc cytochrome crystals of about 5 microm are also investigated by synchrotron radiation diffraction in order to attempt a preliminary analysis of the atomic structure of this unique protein system yet unsolved.

Cholesterol Side-Chain Cleavage Enzyme↗
PubMed2006

Structure and growth of ultrasmall protein microcrystals by synchrotron radiation: II. microGISAX and microscopy of lysozyme.

The early steps of growth and nucleation of the lysozyme microcrystals by classical and nanotemplate-based hanging vapor diffusion methods are studied using microGISAXS at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. Out-of-plane cuts in the Yoneda regions of the 2D scattering profiles point to the detection of ultrasmall lysozyme crystals by microGISAXS quite before than by light microscopy. Furthermore lysozyme crystal formation occurs quite earlier with the nanotemplate than with the classical method. Our data are compatible with two distinct modes of crystal nucleation and growth for P450sc and lysozyme.

Crystallization↗
PubMed2006

Mapping electrostatic potential of a protein on its hydrophobic surface: implications for crystallization of Cytochrome P450scc.

Calculation and combined visualization of electrostatic and hydrophobic properties of Cytochrome P450scc based on two very different homology models allowed to identify extensive hydrophobic patches with neutral electrostatic potential and mutations removing such patches and thus expecting to facilitate crystallization of Cytochrome P450scc, especially for the nanotemplate crystallization method. Implications are discussed for optimizing crystallization and other aspects of protein surface properties and protein recognition.

Animals↗
PubMed2006

Nanostructured biofilms and biocrystals.

The design, the physical properties and the implementation of nanostructured protein biofilms and nanostructured biocrystals are here summarized from ab initio considerations. Particular emphasis is placed on those being developed in our Institute having unique properties useful in a wide range of applications.

Animals↗
PubMed2005

microGISAXS and protein nanotemplate crystallization: methods and instrumentation.

Microbeam grazing-incidence small-angle X-ray scattering (microGISAXS) has been used and the technique has been improved in order to investigate protein nucleation and crystal growth, assisted by a protein nanotemplate. The aim is to understand the protein nanotemplate method in detail, as this method has been proved capable of accelerating and increasing crystal size and quality as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing lysozyme crystals at three different stages of growth.

Crystallography, X-Ray↗
PubMed2005

Investigating crystal-growth mechanisms with and without LB template: protein transfer from LB to crystal.

Protein nanocrystallography has recently been introduced as a unique nanotechnology-based approach to forming stable protein crystals and to characterize them down to atomic resolution. In particular, a protein nanostuctured template appears to be capable of stimulating nucleation and crystal growth of so far unsolved proteins. In the present communication, aimed at investigating the lysozyme crystal-growth mechanisms with and without nanotemplate, the lysozymes appear to transfer directly from the nanostructured film into the drop to trigger the formation of the crystal, therefore highlighting the physical interpretation of the mechanism for nanotemplate-induced protein crystallization.

Animals↗
PubMed2005

Comparison of lysozyme structures derived from thin-film-based and classical crystals.

The present report is dedicated to a systematic comparison of crystal structures produced by the nanobiofilm template method and by the classical hanging-drop vapour-diffusion method. Crystals grown by the innovative nanostructured template method appear indeed radiation-resistant even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The implications of this finding for protein crystallography are discussed here in terms of water redistribution and of the detailed atomic resolution comparative studies of the two crystal structures with or without nanobiofilm template, as emerging also from circular-dichroism and thermal denaturation studies.

Animals↗
PubMed2004

Atomic structure of a CK2alpha human kinase by microfocus diffraction of extra-small microcrystals grown with nanobiofilm template.

Extra-small microcrystals of a human kinase CK2alpha were obtained for the first time by the optimization of a recent protein crystallization method based on highly packed protein nanofilm template. Protein crystal induction and growth appear indeed optimal at high surface pressure of the film template yielding high protein orientation and packing. The resulting extra-small CK2alpha microcrystals (of about 20 microm in diameter) was subsequently used for synchrotron radiation diffraction data collection, which proves possible by means of the Microfocus Beamline at the ESRF Synchrotron in Grenoble. The quality of the resulting crystal diffraction patterns and of its resulting atomic structure at 2.4 A resolution proves the unique validity of the above two combined frontier technologies in defining a new approach to structural proteomics capable to solve the atomic structure of proteins so far never been crystallized and of pharmaceutical relevance. Physical explanation in terms of template dipole moments and possibility of generalization of this method to the wide class of proteins not yet crystallized are finally discussed. The structure of our CK2alpha mutant is in the Protein Data Bank (PDB ID Code 1NA7, deposited on 27 November 2002).

Air Pressure↗
PubMed2004

Protein nanocrystallography: a new approach to structural proteomics.

This article describes a new approach to structural proteomics that can produce and characterize diffracting, stable and radiation-resistant crystals of miniscule dimensions using nanotechnology. We believe that the protein microcrystals obtained by nanotechnology-based protein thin-film template crystallization, as well as groundbreaking technology, such as atomic force microscopy, nanogravimetry and synchrotron microfocus, have enabled protein nanocrystallography to be defined as a unique technology capable of forming and characterizing stable protein microcrystals down to atomic resolution. A new route from art to science and technology has, therefore, been opened in protein crystallography, and it could be used to unravel the mysteries of many systems that remain unsolved.

Casein Kinase II↗
PubMed2003

Three-dimensional atomic structure of a catalytic subunit mutant of human protein kinase CK2.

The three-dimensional crystal structure of the triple-point mutant of the catalytic subunit of human protein kinase CK2alpha has been determined at 2.4 A resolution. Microcrystals of mutant CK2 catalytic subunit were obtained by a protein-crystallization method based on thin-film nanotechnology. These microcrystals (of about 20 micro m in diameter) were used for diffraction data collection by means of the microfocus beamline at the ESRF synchrotron. A comparison between the human protein kinase CK2alpha and the corresponding enzyme from a lower organism (Zea mays) is made.

Amino Acid Sequence↗
PubMed2002

Protein nucleation and crystallization by homologous protein thin film template.

A new method of protein nucleation and crystallization based on Langmuir-Blodgett technology is here utilized for the template stimulation of crystal growth of so far non-crystallized proteins. Microcrystals (60-120 microm) of bovine cytochrome P450scc and human protein kinase CKII alpha subunit were obtained with use of the homologous protein thin film template by vapor diffusion modified hanging drop method. The induction of microcrystals nucleation by the thin template confirms in the two different important classes of proteins, until now never crystallized, the positive stimulatory influence for crystal formation of protein thin film template, which was observed in an earlier study with a model system (chicken egg white lysozyme) as an unexpected acceleration and enhancement in the crystal growth.

Animals↗
PubMed2002

DNASER II: novel surface patterning for biomolecular microarray.

A new matrix-integral part of the new DNA microarray instrumentation DNA analyzer (DNASER) is introduced based on a novel DNA patterning on the solid support surface. Such patterning found the way to modify a glass surface for a precise positioning of small droplets of aqueous DNA solutions, without special robots (arrayers), within the boundaries of the modified regions. The physically heterogeneous surface consists of highly hydrophilic spots surrounded by a highly hydrophobic area leading to the surface patterning needed for a DNA microarray: a matrix of hydrophilic spots properly activated for immobilization of oligonucleotides has been fabricated on absolutely passive hydrophobic surface. The optimal efficiency of the above functionalitation technology of a glass-substrate in obtaining DNA microarray was confirmed by the Cy3-dCTP-labeled DNA sample, as shown by charge-coupled device images of the DNASER previously described.

Biocompatible Materials↗