Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.     

AT excursion: a new approach to predict replication origins in viral genomes by locating AT-rich regions

Background  

Replication origins are considered important sites for understanding the molecular mechanisms involved in DNA replication. Many computational methods have been developed for predicting their locations in archaeal, bacterial and eukaryotic genomes. However, a prediction method designed for a particular kind of genomes might not work well for another. In this paper, we propose the AT excursion method, which is a score-based approach, to quantify local AT abundance in genomic sequences and use the identified high scoring segments for predicting replication origins. This method has the advantages of requiring no preset window size and having rigorous criteria to evaluate statistical significance of high scoring segments.     

Proteomic response of rice seedling leaves to elevated CO2 levels

Previous investigations of plant responses to higher CO 2 levels were mostly based on physiological measurements and biochemical assays. In this study, a proteomic approach was employed to investigate plant response to higher CO 2 levels using rice as a model. Ten-day-old seedlings were progressively exposed to 760 ppm, 1140 ppm, and 1520 ppm CO 2 concentrations for 24 h each. The net photosynthesis rate ( P n), stomatal conductance ( G s), transpiration rate ( E), and intercellular to ambient CO 2 concentration ratio ( C i/ C a) were measured. P n, G s, and E showed a maximum increase at 1140 ppm CO 2, but further exposure to 1520 ppm for 24 h resulted in down regulation of these. Proteins extracted from leaves were subjected to 2-DE analysis, and 57 spots showing differential expression patterns, as detected by profile analysis, were identified by MALDI-TOF/TOF-MS. Most of the proteins belonged to photosynthesis, carbon metabolism, and energy pathways. Several molecular chaperones and ascorbate peroxidase were also found to respond to higher CO 2 levels. Concomitant with the down regulation of P n and G s, the levels of enzymes of the regeneration phase of the Calvin cycle were decreased. Correlations between the protein profiles and the photosynthetic measurements at the three CO 2 levels were explored.     

Permeation of ions across the potassium channel: brownian dynamics studies

The physical mechanisms underlying the transport of ions across a model potassium channel are described. The shape of the model channel corresponds closely to that deduced from crystallography. From electrostatic calculations, we show that an ion permeating the channel, in the absence of any residual charges, encounters an insurmountable energy barrier arising from induced surface charges. Carbonyl groups along the selectivity filter, helix dipoles near the oval chamber, and mouth dipoles near the channel entrances together transform the energy barrier into a deep energy well. Two ions are attracted to this well, and their presence in the channel permits ions to diffuse across it under the influence of an electric field. Using Brownian dynamics simulations, we determine the magnitude of currents flowing across the channel under various conditions. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength causes a steady decrease in the channel conductance. The current also decreases systematically when the effective dielectric constant of the channel is lowered. The conductance with the optimal choice of dipoles reproduces the experimental value when the dielectric constant of the channel is assumed to be 60. The current-voltage relationship obtained with symmetrical solutions is linear when the applied potential is less than approximately 100 mV but deviates from Ohm's law at a higher applied potential. The reversal potentials obtained with asymmetrical solutions are in agreement with those predicted by the Nernst equation. The conductance exhibits the saturation property observed experimentally. We discuss the implications of these findings for the transport of ions across the potassium channels and membrane channels in general.     

Novel fluorescent protein from Hydnophora rigida possesses green emission

Fluorescent proteins are a family of proteins capable of producing fluorescence at various specific wavelengths of ultra violet light. We have previously reported the identification and characterization of a novel cyan fluorescent protein (HriCFP) from a reef coral species, Hydnophora rigida. In search of new members of the diverse family of fluorescent proteins, here we report a new green fluorescent protein (HriGFP) from H. rigida. HriGFP was identified, cloned, expressed in Escherichia coli and purified to homogeneity by metal affinity and size exclusion chromatography. The dynamic light scattering and gel filtration experiments suggested the presence of monomers in solution. The peptide mass fingerprint on the purified protein established the identity of HriGFP. HriGFP had excitation peak at 507 nm and emission peak at 527 nm. HriGFP was similar to HriCFP except the last 16 amino acid sequence at the C-terminal; however, they have shown least similarity with other known fluorescent proteins. Moreover the computational model suggests that HriGFP is a globular protein which consists of 6 α-helices and 3 β-sheets. Taken together our results suggested that HriGFP is a novel naturally occurring fluorescent protein that exists as a monomer in solution.     

Naturally zinc-containing bacteriochlorophyll a ([Zn]-BChl a) protects the photosynthetic apparatus of Acidiphilium rubrum from copper toxicity damage

In almost all photosynthetic organisms the photosynthetic pigments chlorophyll and bacteriochlorophyll (BChl) are Mg²⁺ containing complexes, but Mg²⁺ may be exchanged against other metal ions when these are present in toxic concentrations, leading to inactivation of photosynthesis. In this report we studied mechanisms of copper toxicity to the photosynthetic apparatus of Acidiphilium rubrum, an acidophilic purple bacterium that uses Zn²⁺ instead of Mg²⁺ as the central metal in the BChl molecules ([Zn]-BChl) of its reaction centres (RCs) and light harvesting proteins (LH1). We used a combination of in vivo measurements of photosynthetic activity (fast fluorescence and absorption kinetics) together with analysis of metal binding to pigments and pigment-protein complexes by HPLC-ICP-sfMS to monitor the effect of Cu²⁺ on photosynthesis of A. rubrum. Further, we found that its cytoplasmic pH is neutral. We compared these results with those obtained from Rhodospirillum rubrum, a purple bacterium for which we previously reported that the central Mg²⁺ of BChl can be replaced in vivo in the RCs by Cu²⁺ under environmentally realistic Cu²⁺ concentrations, leading to a strong inhibition of photosynthesis. Thus, we observed that A. rubrum is much more resistant to copper toxicity than R. rubrum. Only slight changes of photosynthetic parameters were observed in A. rubrum at copper concentrations that were severely inhibitory in R. rubrum and in A. rubrum no copper complexes of BChl were found. Altogether, the data suggest that [Zn]-BChl protects the photosynthetic apparatus of A. rubrum from detrimental insertion of Cu²⁺ (trans-metallation) into BChl molecules of its RCs.     

Detection of the patulin-producing potential of some <Emphasis Type="Italic">Paecilomyces variotii</Emphasis> strains isolated from the air samples of Jeddah City,Saudi Arabia,using the RAPD-PCR technique

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis was used to detect the patulin-producing potential among seven different strains of Paecilomyces variotii isolated from air samples collected in Jeddah City, Saudi Arabia. Using five different primers, the strains showed some similarities and distinct RAPD patterns. A correlation between isolation source and clustering was noted in the constructed dendrogram. Patulin-producing strains showed identical RAPD patterns. Primer M13 produced a distinct fragment with toxin-producing strains.     

Complex inheritance of the plasmodial surface anion channel in a Plasmodium falciparum genetic cross

Human erythrocytes infected with the malaria parasite Plasmodium falciparum have increased permeabilities to many solutes. The plasmodial surface anion channel (PSAC) may mediate these changes. Despite good understanding of the biochemical and biophysical properties, the genetic basis of PSAC activity remains unknown. Functional polymorphisms in laboratory isolates and two mutants generated by in vitro selection implicate a parasite-encoded channel, although parasite-induced modifications of endogenous channels have not been formally excluded. Here, we identified stable differences in furosemide efficacy against PSAC activity induced by HB3 and 3D7A parasites. This difference was apparent in both single PSAC patch-clamp recordings and in sorbitol-mediated osmotic lysis measurements, confirming that Cl^- and sorbitol are transported by a single-channel type. Examination of 19 progeny from a genetic cross between HB3 and 3D7A revealed complex inheritance with some cloned progeny exhibiting furosemide affinities outside the range of parental values. Isolates generated by selfing of the 3D7A clone also exhibited altered furosemide affinities, implicating changes in one or more alleles during meiosis or passage through a primate host. PSAC may be encoded by multiple parasite genes (e.g. a multi-gene family or multiple genes that encode distinct channel subunits) or a single polymorphic gene under strong selective pressure.     

Purification and characterization of xylanases from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> and its mutant derivative possessing novel kinetic and thermodynamic properties

Xylanases produced from a locally isolated strain of Thermomyces lanuginosus and its mutant derivative were purified to a yield of 39.1 and 42.83% with specific activities of 15,501 and 17,778 IU mg⁻¹ protein, respectively. The purification consisted of two steps i.e., ammonium sulphate precipitation, and gel filtration chromatography. The mutant enzyme showed high affinity for substrate, with a K _m of 0.098 mg ml⁻¹ as compared to wild type enzyme showing K _m of not less than 0.112 mg ml⁻¹. It was found that pH values of 8.1 and 7.3 were best for activity of the mutant and wild-type-derived enzymes, respectively. The values of pK _a of the acidic limbs of both enzymes were the same (5.0 and 4.9, respectively) but the pK _a value of the basic limb was slightly increased, indicating the participation of a carboxyl group present in a non-polar environment. Temperatures of 70 and 65°C were found optimal for mutant and wild-derived xylanase, respectively. Enzymes displayed a high thermostability showing a half life of 31.79 and 6.0 min (5.3-fold improvement), enthalpy of denaturation (ΔH*) of 146.06 and 166.95 kJ mol⁻¹, entropy of denaturation (ΔS*) of 101.44 and 174.67, and free energy of denaturation (ΔG*) of 110.25 and 105.29 kJ mol⁻¹ for mutant- and wild-organism derived enzyme, respectively at 80°C. Studies on the folding and stability of cellulase-less xylanases are important, since their biotechnological employments require them to function under extreme conditions of pH and temperature. The kinetic and thermodynamic properties suggested that the xylanase from the mutant organism is better as compared to xylanase produced from the wild type and previously reported strains of same species, and may have a potential usage in various industrial fields.