Biomedical subjects

Olga Tcherkasskaya

Publications and source records attributed to Olga Tcherkasskaya.

8 recordsLinked to original sources

PubMed2006

Local flexibility in molecular function paradigm.

It is generally accepted that the functional activity of biological macromolecules requires tightly packed three-dimensional structures. Recent theoretical and experimental evidence indicates, however, the importance of molecular flexibility for the proper functioning of some proteins. We examined high resolution structures of proteins in various functional categories with respect to the secondary structure assessment. The latter was considered as a characteristic of the inherent flexibility of a polypeptide chain. We found that the proteins in functionally competent conformational states might be comprised of 20-70% flexible residues. For instance, proteins involved in gene regulation, e.g. transcription factors, are on average largely disordered molecules with over 60% of amino acids residing in "coiled" configurations. In contrast, oxygen transporters constitute a class of relatively rigid molecules with only 30% of residues being locally flexible. Phylogenic comparison of a large number of protein families with respect to the propagation of secondary structure illuminates the growing role of the local flexibility in organisms of greater complexity. Furthermore the local flexibility in protein molecules appears to be dependent on the molecular confinement and is essentially larger in extracellular proteins.

Arabidopsis↗

PubMed2005

Conformational biosensor for diagnosis of prion diseases.

A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-beta-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.

Amyloid↗

PubMed2004

Recombinant EWS-FLI1 oncoprotein activates transcription.

The Ewing's sarcoma family of tumors (ESFT) contains a characteristic translocation the chimeric transcript of which is translated to become the EWS-FLI1 fusion protein. EWS-FLI1 regulates transcription and posttranscriptional splicing. Elimination of EWS-FLI1 protein from ESFT cells induces apoptosis and reduces xenograft tumor growth. Therefore the production of a biologically active recombinant EWS-FLI1 could lead to discoveries that would enhance our mechanistic understanding of ESFT. We have cloned, expressed, and purified a biologically active recombinant EWS-FLI1 in Escherichia coli using affinity column chromatography. A refolding procedure was required to render the recombinant EWS-FLI1 soluble in relatively native conditions. The structural alterations induced by the refolding procedure were monitored by SDS-gel electrophoresis, circular dichroism, and steady-state fluorescence spectroscopy. Recombinant EWS-FLI1 under native conditions approaches a largely unfolded conformation. Recombinant EWS-FLI1 protein under native conditions specifically binds to DNA and transcribes RNA. Our biologically active recombinant EWS-FLI1 oncoprotein will be useful to identify functional molecular partners and inhibitors.

Amino Acid Sequence↗

PubMed2003

The role of hydrophobic interactions in amyloidogenesis: example of prion-related polypeptides.

Conversion of the non-infectious, cellular form of the prion protein (PrP(C)) to the infectious form (PrP(Sc)) is thought to be driven by an alpha-helical to beta-sheet conformational transition. To reveal the sequence determinants which encourage the transition to beta-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and steady state fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant beta-strand formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichroism. At moderate concentrations of trifluoroethanol and/or acetonitrile, the conformational ensemble shifts towards beta-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid beta-sheet structures. Encouragement of beta-folds requires the destabilization of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms.

Amino Acid Sequence↗

PubMed2003

Polymeric aspects of protein folding: a brief overview.

Regardless of the differences in primary amino acid sequences, protein molecules in a number of conformational states behave as polymer homologues, allowing speculations as to the volume interactions being a driving force in formation of equilibrium structures. For instance, both native and molten globules exhibit key features of polymer globules, where the fluctuations of the molecular density are expected to be much less than the molecular density itself. Protein molecules in the compact denatured (pre-molten globule) states possess properties of squeezed coils. In fact, even high concentrations of strong denaturants (e.g., urea and GdmCl) more likely constitute bad solvents for protein chains. Thus, globular proteins are probably never random coils without positional correlations and biological polypeptide chains represent the macromolecular coils below a critical point even under harsh denaturing conditions. Several implications of these findings to protein folding are discussed.

Animals↗

PubMed2002

Excluded volume effect within the continuous model for the fluorescence energy transfer.

We consider and discuss the transfer of electronic energy between donor and acceptor molecules, both continuously distributed in an infinite space. In particular, the ensemble-average fluorescence intensity decay for the donor was calculated, taking into account the excluded volume. The latter may be associated either with finite molecular size or any other spatial restrictions, which are imposed on fluorophore distribution by a superstructure. Results show that in a system using excluded volume, the time dependence in donor decay is more complex compared to that predicted by a simplified stretched exponential model. We identify a crossover between two distinct time regimes in the refined decay and demonstrate its correlation with two competing parameters: r(m), which characterizes the minimal distance between interacting molecules, and R(0), which is related to the strength of the molecular interactions. In this context, the "apparent dimensionality" of the energy transfer recovered from the stretched exponential model ignores the crossover, and may be quite misleading. Basic theoretical considerations to that end are provided.

Biophysical Phenomena↗

PubMed2002

Effective lattice behavior of fluorescence energy transfer at lamellar macromolecular interfaces.

Fluorescence energy transfer between donors and acceptors confined to macromolecular interfaces is considered. In particular, we discuss two theoretical models for the ensemble-average fluorescence intensity decay of the donor when both fluorophores are incorporated into a planar (e.g., lamellar) interface. The first model is based on a continuous distribution of donor and acceptor molecules on a two-dimensional surface, whereas the other assumes a discrete distribution of fluorophores along the nodes of a two-dimensional square lattice. Results for the discrete model show that the fluorescence intensity kinetics of a donor depends strongly on the geometry of the molecular distribution (i.e., the lattice constant) and the photophysics of fluorophores (i.e., critical radius of the energy transfer). Furthermore, a "discrete molecular distribution" might manifest itself in the experimental data as an increase in the apparent dimensionality of the energy transfer with increasing acceptor concentration. Altogether, the experimental and theoretical underpinnings indicate the enormous potential of using fluorescence energy-transfer kinetics for revealing structural features of molecular ensembles (i.e., geometry, shape) based on a single experimental measurement. However, further understanding the effects of restricted geometries on the fluorescence energy transfer is required to take full advantage of this information. Basic theoretical considerations to that end are provided.

Biophysical Phenomena↗

PubMedDate not supplied

Biophysical constraints for protein structure prediction.

Though highly desirable, neither a single experimental technique nor a computational approach can be sufficient enough to rationalize a protein structure. The incorporation of biophysical constraints, which can be rationalized based on conventional biophysical measurements, might lead to considerable improvement of the simulation procedures. In this regard, our analysis of 180 proteins in different conformational states allows prediction of the overall protein dimension based on the chain length, i.e., the protein molecular weight, with an accuracy of 10%.

Algorithms↗