Local polarity and hydrogen bonding inside the Sec14p phospholipid-binding cavity: high-field multi-frequency electron paramagnetic resonance studies

Sec14p promotes the energy-independent transfer of either phosphatidylinositol (PtdIns) or phosphatidylcholine (PtdCho) between lipid bilayers in vitro and represents the major PtdIns/PtdCho transfer protein in the budding yeast Saccharomyces cerevisiae. Herein, we employ multi-frequency high-field electron paramagnetic resonance (EPR) to analyze the electrostatic and hydrogen-bonding microenvironments for series of doxyl-labeled PtdCho molecules bound by Sec14p in a soluble protein-PtdCho complex. A structurally similar compound, 5-doxyl stearic acid dissolved in a series of solvents, was used for experimental calibration. The experiments yielded two-component rigid limit 130- and 220-GHz EPR spectra with excellent resolution in the gx region. Those components were assigned to hydrogen-bonded and nonhydrogen-bonded nitroxide species. Partially resolved 130-GHz EPR spectra from n-doxyl-PtdCho bound to Sec14p were analyzed using this two-component model and allowed quantification of two parameters. First, the fraction of hydrogen-bonded nitroxide species for each n-doxyl-PtdCho was calculated. Second, the proticity profile along the phospholipid-binding cavity of Sec14p was characterized. The data suggest the polarity gradient inside the Sec14p cavity is a significant contributor to the driving molecular forces for extracting a phospholipid from the bilayer. Finally, the enhanced g-factor resolution of EPR at 130 and 220 GHz provides researchers with a spectroscopic tool to deconvolute two major contributions to the x-component of the nitroxide g-matrix: hydrogen-bond formation and local electrostatic effects.     

Laying characteristics of one- and two-year old pheasants (Phasianus colchicus, L.)

The aim ofthe study was to assess laying traits, the weight of eggs and characters ofthe laying rhythm of pheasants in the first and second years of reproduction. Pheasants (10 cockerels and 50 hens) were kept in aviaries. Daily, individual control of laying was performed beginning with the day of the first laying and ending with the last egg. The following parameters were evaluated: age at first laying, length of the laying period, number of laid eggs and the average weight of the egg in the 8th week of laying. The laying rhythm was also assessed and comprised: the number of egg clutches, the number of eggs in a clutch, the number of eggs in the longest clutch, the number of intervals, the length of intervals and the longest interval between clutches. During the first period of reproduction, in comparison with the second, pheasants laid slightly more eggs of similar average weight. The first laying period was longer than the second and was characterised by a greater number of egg clutches and greater number of intervals between clutches. The greatest number of eggs was laid in 10-egg and longer clutches, although the l-egg clutches were the most numerous. A positive correlation was found between the number of eggs and the number of clutches, the greatest number of eggs in a clutch and the number of intervals between clutches. The similar values of the reproductive characters of one- and two-year old pheasants point to the possibility of longer utilization of these birds than only for one laying period. On the other hand, the considerable variability between the experimental hens with regard to the number and the length of egg clutches, as well as the intervals between them, indicate the possibility to carry out selection taking into account traits characterising the laying rhythm.     

Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA^Lys3 having 2-thiouridine (s²U₃₄) and pseudouridine (Ψ₃₉), bound the modified human ASL^Lys3(s²U₃₄;Ψ₃₉) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL^Lys3(s²U₃₄;Ψ₃₉), followed by the hypermodified ASL^Lys3 (mcm⁵s²U₃₄;ms²t⁶A₃₇) and the unmodified ASL^Lys3, but bound poorly to singly modified ASL^Lys3 constructs (Ψ₃₉, ms²t⁶A_37, s²U₃₄), ASL^Lys1,2 (t⁶A₃₇) and Escherichia coli ASL^Glu (s²U₃₄). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.     

Non-empirical study of the phosphorylation reaction catalyzed by 4-methyl-5-β-hydroxyethylthiazole kinase: relevance of the theory of intermolecular interactions

The subject of this study was an analysis of the role of active site residues in the phosphoryl transfer reaction catalyzed by 4-methyl-5-β-hydroxyethylthiazole kinase (ThiK). The ThiK-catalyzed reaction is of special interest due to the lack of a highly conserved aspartate residue serving as a catalytic base. ONIOM(B3LYP:PM3) models of stationary points along the reaction pathway consisted of reactants, two magnesium ions and several highly conserved ThiK active site residues. The results indicate that an S_N2-like mechanism of ThiK, with γ-phosphate acting as an alcohol-activating base is reasonable. Geometries of substrates, transition state and products were utilized in the non-empirical analysis of the physical nature of catalytic interactions taking place in the ThiK active site. The role of particular residues was investigated in terms of their ability to preferentially stabilize the transition state relative to substrates (differential transition state stabilization, DTSS) or products (differential product stabilization, DPS). It seems that Mg2, Glu126 and Cys198 play a major catalytic role, whereas Mg1 and the same Cys198 are responsible for product release. It is remarkable that no dominant role of an electrostatic term in the interactions involved in catalytic activity is observed for product release. Determination of catalytic fields expressing differential electrostatic potential of the transition state with respect to substrates revealed the optimal electrostatic features of an ideal catalyst for the studied reaction. The predicted catalytic environment is in agreement with experimental data showing increased catalytic activity of ThiK upon mutation of Cys198 to aspartate. Figure Catalytic fields for ThiK-catalyzed reaction juxtaposed with the positions of active site residues of a model system. Magnesium ions are considered part of the transition state/reactants. The surface of constant electronic density is colored according to differential electrostatic potential of transition state with respect to reactants. The sign of the differential potential reflects the electrostatic properties of a complementary molecular environment. Red (green) color denotes regions where a negative (positive) charge would be optimal for catalytic activity     

Air pollution distribution patterns in the San Bernardino Mountains of southern California: a 40-year perspective

Since the mid-1950s, native pines in the San Bernardino Mountains (SBM) in southern California have shown symptoms of decline. Initial studies in 1963 showed that ozone (O3) generated in the upwind Los Angeles Basin was responsible for the injury and decline of sensitive trees. Ambient O3 decreased significantly by the mid-1990s, resulting in decreased O3 injury and improved tree growth. Increased growth of trees may also be attributed to elevated atmospheric nitrogen (N) deposition. Since most of the N deposition to mixed conifer forest stands in the SBM results from dry deposition of nitric acid vapor (HNO3) and ammonia (NH3), characterization of spatial and temporal distribution of these two pollutants has become essential. Although maximum daytime O3 concentrations over last 40 years have significantly decreased (approximately 3-fold), seasonal means have been reduced much less (approximately 1.5-fold), with 2-week long means occasionally exceeding 100 ppb in the western part of the range. In the same area, significantly elevated concentrations of HNO3 and NH3, up to 17.5 and 18.5 microg/m3 as 2-week averages, respectively, have been determined. Elevated levels of O3 and increased N deposition together with long-term drought predispose the SBM forests to massive bark beetle attacks making them susceptible to catastrophic fires.     

Adjoint systems for models of cell signaling pathways and their application to parameter fitting

The paper concerns the problem of fitting mathematical models of cell signaling pathways. Such models frequently take the form of sets of nonlinear ordinary differential equations. While the model is continuous in time, the performance index used in the fitting procedure involves measurements taken at discrete time moments. Adjoint sensitivity analysis is a tool which can be used for finding the gradient of a performance index in the space of parameters of the model. In the paper, a structural formulation of adjoint sensitivity analysis called the generalized backpropagation through time (GBPTT) is used. The method is especially suited for hybrid, continuous-discrete time systems. As an example, we use the mathematical model NF-kappaB of the regulatory module, which plays a major role in the innate immune response in animals.     

In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Aging: all in the head?

Global reduction in insulin signaling has been linked to extended life span in a range of organisms. New work on mice with brain-specific or whole-body reductions in insulin receptor substrate 2 (IRS2) (Taguchi et al., 2007) points to a role for insulin/IGF-1 signaling in the central control of mammalian aging.