The Denaturant- and Mutation-Induced Disassembly of Pseudomonas aeruginosa Hexameric Hfq Y55W Mutant.

Although oligomeric proteins are predominant in cells, their folding is poorly studied at present. This work is focused on the denaturant- and mutation-induced disassembly of the hexameric mutant Y55W of the Qß host factor (Hfq) from mesophilic Pseudomonas aeruginosa (Pae). Using intrinsic tryptophan fluorescence, dynamic light scattering (DLS), and high-performance liquid chromatography (HPLC), we show that the dissociation of Hfq Y55W occurs either under the effect of GuHCl or during the pre-denaturing transition, when the protein concentration is decreased, with both events proceeding through the accumulation of stable intermediate states. With an extremely low pH of 1.4, a low ionic strength, and decreasing protein concentration, the accumulated trimers and dimers turn into monomers. Also, we report on the structural features of monomeric Hfq resulting from a triple mutation (D9A/V43R/Y55W) within the inter-subunit surface of the protein. This globular and rigidly packed monomer displays a high thermostability and an oligomer-like content of the secondary structure, although its urea resistance is much lower.

The major mRNP protein YB-1: structural and association properties in solution.

YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional (1)H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.

A comparative analysis of lecturers' satisfaction with Anatomage and Pirogov virtual dissection tables during clinical and topographic anatomy courses in Russian universities.

Anatomy is increasingly taught using computer-assisted learning tools, including electronic interactive anatomy dissection tables. Anatomage was he first virtual anatomy dissection table introduced in Russian medical universities and gained popularity among lecturers and students. The Pirogov interactive anatomy table was recently released, but the strengths and weakness of each platform is currently unknown. The objective of this article is to survey lecturers in anatomy to understand their perspectives on the Pirogov versus Anatomage virtual dissection tables' application to teaching in medical universities. A total of 80 anatomy educators from 12 Russian universities, using Anatomage (n = 40) and Pirogov (n = 40) tables were surveyed regarding their satisfaction with the application of the respective tables. Using a five-point Likert scale, both tables were assessed, and responses were statistically analyzed. In addition, qualitative analysis was performed on free response comments provided by survey respondents. There was no significant difference in overall satisfaction ratings between Pirogov (4.38 ± 0.53) and Anatomage (3.94 ± 0.60) interactive tables (p > 0.05). The Anatomage table ranked significantly higher on the accuracy of displayed anatomical details, resolution of the images, and its suitability for teaching senior medical and postgraduate students. Pirogov table performed significantly better on survey items measuring ergonomics, ability to assess students' performance, and teaching basic anatomy to junior first- and second-year medical students. Thus, in summary, anatomists' responses indicated that while both tables are suitable for teaching anatomy, the Pirogov table was superior in undergraduate medical education and the Anatomage table was more beneficial for teaching more senior trainees.

Recombinant Globular Domain of TcpA Pilin from Vibrio cholerae El Tor: Recovery from Inclusion Bodies and Structural Characterization.

The production of recombinant proteins in Escherichia coli cells is often hampered by aggregation of newly synthesized proteins and formation of inclusion bodies. Here we propose the use of transverse urea gradient electrophoresis (TUGE) in testing the capability of folding of a recombinant protein from inclusion bodies dissolved in urea. A plasmid encoding the amino acid sequence 55-224 of TcpA pilin (C-terminal globular domain: TcpA-C) from Vibrio cholerae El Tor enlarged by a His-tag on its N-terminus was expressed in E. coli cells. The major fraction (about 90%) of the target polypeptide was detected in cell debris. The polypeptide was isolated from the soluble fraction and recovered from inclusion bodies after their urea treatment. Some structural properties of the polypeptide from each sample proved identical. The refolding protocol was developed on the basis of TUGE data and successfully used for the protein large-scale recovery from inclusion bodies. Spectral, hydrodynamic, and thermodynamic characteristics of the recombinant TcpA recovered from inclusion bodies indicate the presence of a globular conformation with a pronounced secondary structure and a rigid tertiary structure, which is promising for the design of immunodiagnostics preparations aimed to assess the pilin level in different strains of V. cholerae and to develop cholera vaccines.

Solution structure and dynamics of the chimeric SH3 domains, SHH- and SHA-"Bergeracs".

Two chimeric proteins, SHcapital EN, Cyrillic and SHA of the "SH3-Bergerac" family (where the beta-turn N47D48 in spectrin SH3 domain was substituted for KITVNGKTYE or KATANGKTYE sequences, respectively), were analyzed by high-resolution NMR to resolve their spatial structures and to analyze their dynamics. Although the presence of a stable beta-hairpin in the region of the insertion was confirmed, the introduced extension of the polypeptide chain in SHcapital EN, Cyrillic (approximately 17%) practically did not affect the total molecule topology. Interestingly, the introduced beta-hairpin had higher mobility in comparison with other protein regions. Finally, we performed a disorder prediction with the PONDR VSL2 algorithm and discovered that the inserted beta-hairpin in both SHH and SHA proteins exhibited significant propensity for intrinsic disorder and therefore for high mobility. In agreement with the experimental data, the predisposition for the increased intramolecular mobility was noticeably higher in SHA.

Detection of intensity bursts using Hawkes processes: An application to high-frequency financial data.

Given a stationary point process, an intensity burst is defined as a short time period during which the number of counts is larger than the typical count rate. It might signal a local nonstationarity or the presence of an external perturbation to the system. In this paper we propose a procedure for the detection of intensity bursts within the Hawkes process framework. By using a model selection scheme we show that our procedure can be used to detect intensity bursts when both their occurrence time and their total number is unknown. Moreover, the initial time of the burst can be determined with a precision given by the typical interevent time. We apply our methodology to the midprice change in foreign exchange (FX) markets showing that these bursts are frequent and that only a relatively small fraction is associated with news arrival. We show lead-lag relations in intensity burst occurrence across different FX rates and we discuss their relation with price jumps.

The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride.

The unfolding thermodynamics of the circular enterocin protein AS-48, produced by Enterococcus faecalis, has been studied. The native structure of the 70-amino-acid-long protein turned out to be extremely stable against heat and denaturant-induced unfolding. At pH 2.5 and low ionic strength, it denatures at 102 degrees C, while at 25 degrees C, the structure only unfolds in 6.3 M guanidinium hydrochloride (GuHCl) and does not unfold even in 8 M urea. A comparison of its thermal unfolding in water and in the presence of urea shows a good correspondence between the two deltaGw(298) values, which are about 30 kJ mol(-1) at pH 2.5 and low ionic strength. The stability of the structure is highly dependent upon ionic strength and so GuHCl acts both as a denaturant and a stabilising agent. This seems to be why the deltaGw(298) value calculated from the unfolding data in GuHCl is twice as high as in the absence of this salt. At least part of the high stability of native AS-48 can almost certainly be put down to its circular organization since other structural features are quite normal for a protein of this size.

A thermodynamic and kinetic analysis of the folding pathway of an SH3 domain entropically stabilised by a redesigned hydrophobic core.

The folding thermodynamics and kinetics of the alpha-spectrin SH3 domain with a redesigned hydrophobic core have been studied. The introduction of five replacements, A11V, V23L, M25V, V44I and V58L, resulted in an increase of 16% in the overall volume of the side-chains forming the hydrophobic core but caused no remarkable changes to the positions of the backbone atoms. Judging by the scanning calorimetry data, the increased stability of the folded structure of the new SH3-variant is caused by entropic factors, since the changes in heat capacity and enthalpy upon the unfolding of the wild-type and mutant proteins were identical at 298 K. It appears that the design process resulted in an increase in burying both the hydrophobic and hydrophilic surfaces, which resulted in a compensatory effect upon the changes in heat capacity and enthalpy. Kinetic analysis shows that both the folding and unfolding rate constants are higher for the new variant, suggesting that its transition state becomes more stable compared to the folded and unfolded states. The phi(double dagger-U) values found for a number of side-chains are slightly lower than those of the wild-type protein, indicating that although the transition state ensemble (TSE) did not change overall, it has moved towards a more denatured conformation, in accordance with Hammond's postulate. Thus, the acceleration of the folding-unfolding reactions is caused mainly by an improvement in the specific and/or non-specific hydrophobic interactions within the TSE rather than by changes in the contact order. Experimental evidence showing that the TSE changes globally according to its hydrophobic content suggests that hydrophobicity may modulate the kinetic behaviour and also the folding pathway of a protein.

Geriatric patients: compliance issues and ways of its optimization

The aim of the study is to increase the effectiveness of diseases treatment in elderly patients. The work is based on the study of the nature and the role of compliance disorders, optimizing the effectiveness with the use of therapeutic complex of pharmacotherapy associated with psychotherapeutic component. As a result of the analysis of morbidity in elderly age and the nature of compliance in elderly patients, an optimal therapeutic complex was proposed, which consists of rational pharmacological load and psychotherapeutic intervention, aimed at informing a patient about the disease, methods of its treatment, as well as at creating an effective therapeutic alliance which provides proper medical care and prevents self-treatment in such patients.

Quantifying reflexivity in financial markets: toward a prediction of flash crashes.

We introduce a measure of activity of financial markets that provides a direct access to their level of endogeneity. This measure quantifies how much of price changes is due to endogenous feedback processes, as opposed to exogenous news. For this, we calibrate the self-excited conditional Poisson Hawkes model, which combines in a natural and parsimonious way exogenous influences with self-excited dynamics, to the E-mini S&P 500 futures contracts traded in the Chicago Mercantile Exchange from 1998 to 2010. We find that the level of endogeneity has increased significantly from 1998 to 2010, with only 70% in 1998 to less than 30% since 2007 of the price changes resulting from some revealed exogenous information. Analogous to nuclear plant safety measures concerned with avoiding "criticality," our measure provides a direct quantification of the distance of the financial market from a critical state defined precisely as the limit of diverging trading activity in the absence of any external driving.

High-resolution crystal structure of spectrin SH3 domain fused with a proline-rich peptide.

A new chimeric protein, named WT-CIIA, was designed by connecting the proline-rich decapeptide PPPVPPYSAG to the C-terminus of the alpha-spectrin SH3 domain through a natural twelve-residue linker to obtain a single-chain model that would imitate intramolecular SH3-ligand interaction. The crystal structure of this fusion protein was determined at 1.7 Å resolution. The asymmetric unit of the crystal contained two SH3 globules contacting with one PPPVPPY fragment located between them. The domains are related by the two-fold non-crystallographic axis and the ligand lies in two opposite orientations with respect to the conservative binding sites of SH3 domains.

Structural and thermodynamic studies of Bergerac-SH3 chimeras.

Bergerac-type chimeras of spectrin SH3 were designed by extending a beta-hairpin by eight amino acids so that the extension protruded from the domain body like a "nose" being exposed to the solvent. A calorimetric study of several Bergerac-SH3 variants was carried out over a wide range of pH values and protein concentrations and the three-dimensional structure of one of them, SHH, was determined. X-ray studies confirmed that the nose had a well defined beta-structure whilst the chimera formed a stable tetramer within the crystal unit because of four tightly packed noses. In the pH range of 4-7 the heat-induced unfolding of some chimeras was complex and concentration dependent, whilst at pH values below 3.5, low protein concentrations of all the chimeras studied, including SHH, seemed to obey a monomolecular two-state unfolding model. The best set of data was obtained for the SHA variant, the unfolding heat effects of which were systematically higher than those of the WT protein (about 16.4 kJ/mol at 323 K), which may be close to the upper limit of the enthalpy gain due to 10 residue beta-hairpin folding. At the same time, the chimeras with high nose stability, which, like SHH, have a hydrophobic (IVY) cluster on their surface, showed a lower apparent unfolding heat effect, much closer to that of the WT protein. The possible reasons for this difference are discussed.

Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant.

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 micro m of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomeric one. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding, whereas the quite small value for the unfolding heat capacity change (3.7 kJ.K(-1).mol(-1)) stabilizes the protein within a broad temperature range, as shown by the stability curves (Gibbs energy functions vs. temperature), even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH 4.00 and 3.75, but becomes considerably less so at pH 3.50 and below, to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degrees C for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.

Stability of Natrialba magadii NDP kinase: comparisons with other halophilic proteins.

Nucleoside diphosphate kinase from the haloalkaliphilic archaeon Natrialba magadii (Nm NDPK) is a homooligomeric hexamer with a monomer molecular weight of 23 kDa. Its main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Previously it was shown that Nm NDPK is active over a wide range of NaCl concentrations, which is not typical of extremely halophilic proteins. In this paper more detailed investigations of kinase function and stability were carried out using circular dichroism, differential scanning calorimetry, size-exclusion chromatography, and biochemical methods. A possible mechanism for stabilization of halophilic proteins that allows them to function in a wide range of NaCl concentrations is proposed.