Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.     

DNA bending due to specific p53 and p53 core domain-DNA interactions visualized by electron microscopy

We have used transmission electron microscopy to analyze the specificity and the extent of DNA bending upon binding of full-length wild-type human tumor suppressor protein p53 (p53) and the p53 core domain (p53CD) encoding amino acid residues 94-312, to linear double-stranded DNA bearing the consensus sequence 5'-AGACATGCCTAGACATGCCT-3' (p53CON). Both proteins interacted with high specificity and efficiency with the recognition sequence in the presence of 50 mM KCl at low temperature ( approximately 4 degrees C) while the p53CD also exhibits a strong and specific interaction at physiological temperature. Specific complex formation did not result in an apparent reduction of the DNA contour length. The interaction of p53 and the p53CD with p53CON induced a noticeable salt-dependent bending of the DNA axis. According to quantitative analysis with folded Gaussian distributions, the bending induced by p53 varied from approximately 40 degrees to 48 degrees upon decreasing of the KCl concentration from 50 mM to approximately 1 mM in the mounting buffer used for adsorption of the complexes to the carbon film surface. The p53CD bent DNA by 35-37 degrees for all salt concentrations used in the mounting buffer. The bending angle of the p53/DNA complex under low salt conditions showed a somewhat broader distribution (sigma approximately 39 degrees ) than at high salt concentration (sigma approximately 31 degrees ) or for p53CD (sigma approximately 24-27 degrees ). Together, these results demonstrate that the p53CD has a dominant role in complex formation and that the complexes formed both by p53 and p53CD under moderate salt conditions are similar. However, the dependence of the bending parameters on ambient conditions suggest that the segments flanking the p53CD contribute to complex formation as well. The problems associated with the analysis of bending angles in electron microscopy experiments are discussed.     

Brownian dynamics simulations of molecular recognition in an antibody-antigen system.

The crystal structure for an antibody-antigen system, that of the anti-hen egg lysozyme monoclonal antibody HyHEL-5 complexed to lysozyme, is used as the starting point for computer simulations of diffusional encounters between the two proteins. The investigation consists of two parts: first, the linearized Poisson-Boltzmann equation is solved to determine the long-range electrostatic forces between antibody and antigen, and then, the relative motion as influenced by these forces is modeled within Brownian motion theory. The effects of various point mutations on the calculated reaction rate are considered. It is found that charged residues close to the binding site exert the greatest influence in steering the proteins into a configuration favorable for their binding, while more distant mutations are qualitatively described by the Smoluchowski model for the mutual diffusion of two uniformly charged spheres. The antibody residues involved in forming salt links with the lysozyme, Glu-H35 and Glu-H50, appear to be particularly important in electrostatic steering, as neutralization of both of them yields reaction rates that are two to three orders of magnitude below those of wild-type rates. The relative rates obtained from the simulations can be tested through kinetic measurements on mutant protein complexes. Kinetically efficient partners can also be designed and constructed through directed mutagenesis.     

Chwaka Bay (Zanzibar,East Africa) as a nursery ground for penaeid prawns

The penaeid prawns are recruited in the sheltered sandy beaches and mangrove areas of Chwaka Bay (Zanzibar) at post-larval stage (7 mm). Year round incursions with a maximum during the warmer months of December to March were observed. February to March is considered as the peak recruitment period. Out of six species of penaeids represented in the area, Penaeus latisulcatus (75%) and Penaeus indicus (15%) were dominant. The recruitment pattern indicated greatest incursions of post-larvae with the flood spring tides of the night when the tidal flow is strong.The juvenile population of P. latisulcatus is distributed in those intertidal sandflats with a rich growth of seagrass and P. indicus in the muddy areas of mangrove forests. P. indicus showed affinity for euryhaline conditions, whereas P. latisulcatus showed no preference for lower salinity. Provision of food and shelter are considered as important factors for their nursery dependence. P. latisulcatus attained a size of 60–70 mm in five to six months and P. indicus 110–120 mm in six to eight months during their nursery phase. These juvenile penaeids were found to be omnivorous, feeding on animal products, plant material and detritus.The maturing P. latisulcatus emigrate back to the sea when they are about 65 mm, whereas P. indicus move out at about 120 mm. A positive correlation between post-larval recruitment and juvenile abundance was observed.     

Long-lived photoinduced charge separation in new Ru(bipyridine)(3)(2+)-phenothiazine dyads

Synthesis and photophysical properties of three Ru(bpy)(3)(2+)-Ptz (bpy = 2,2'-bipyridine and Ptz = phenothiazine) dyads, where the number of Ptz groups increased from one to three, are reported. The MLCT absorption bands of these compounds were slightly red shifted compared to Ru(bpy)(3)(2+). The emission, however, was highly quenched and this is attributed to electron transfer from the Ptz moiety to the excited Ru(bpy)(3)(2+) to generate the charge separated state Ru(bpy)(3)(+)-Ptz (+). Observed electron transfer rates (k(et) > 10(8) s(-1)) were much faster than those previously reported (k(et) < 10(7) s(-1)) for linked Ru(bpy)(3)(2+)-Ptz systems. Compared to the previous systems, back electron transfer rates in these systems were about 100 times slower. This has enabled us to observe the charge separated state in nanosecond flash photolysis experiments. Transient absorptions assignable to Ru(bpy)(3)(+) and Ptz (+), having lifetimes in the range of 10-30 ns were observed. In order to explain the fast charge separation and slow charge recombination rates, formation of a folded conformer where the Ptz group attached to one bpy residue comes closer to and associates with another bpy moiety was invoked. A scheme which explains the fast electron transfer and slow recombination in this pre-associated state is proposed.     

Three components coupling catalysed by NaHSO(4).Sio(2) - a convenient synthesis, antibacterial and antifungal activities of novel 6-Aryl-1,2,4,5-tetrazinan-3-ones

A novel method has been developed for the synthesis of 6-aryl-1,2,4,5-tetrazinan-3-ones through a one-pot reaction of urea, various substituted aromatic benzaldehyde having electron donating and electron withdrawing groups and ammonium acetate in the presence of reusable NaHSO(4).SiO(2) heterogeneous catalyst in dry media under microwave irradiation. FT-IR, (1)H NMR, D(2)O Exchange, (13)C NMR, Heteronuclear Single Quantum Correlation (HSQC) spectra, MS and elemental analysis characterized all the synthesized compounds. In vitro antibacterial/fungal activities were evaluated for six new compounds. The antibacterial studies revealed that compounds 1-6 had better activity against tested Gram-positive and Gram-negative organisms. Compounds 1 and 5 were more active against beta-Heamolytic streptococcus, a Gram-positive bacteria and Pseudomonas, a Gram-negative bacteria, respectively, than the standard drug ciprofloxacin. Besides, of all the compounds tested, compound 5 was more effective against Aspergillus flavus, a fungal strain than the standard drug fluconazole.     

Functional coherence in domain interaction networks

MOTIVATION: Extracting functional information from protein-protein interactions (PPI) poses significant challenges arising from the noisy, incomplete, generic and static nature of data obtained from high-throughput screening. Typical proteins are composed of multiple domains, often regarded as their primary functional and structural units. Motivated by these considerations, domain-domain interactions (DDI) for network-based analyses have received significant recent attention. This article performs a formal comparative investigation of the relationship between functional coherence and topological proximity in PPI and DDI networks. Our investigation provides the necessary basis for continued and focused investigation of DDIs as abstractions for functional characterization and modularization of networks. RESULTS: We investigate the problem of assessing the functional coherence of two biomolecules (or segments thereof) in a formal framework. We establish essential attributes of admissible measures of functional coherence, and demonstrate that existing, well-accepted measures are ill-suited to comparative analyses involving different entities (i.e. domains versus proteins). We propose a statistically motivated functional similarity measure that takes into account functional specificity as well as the distribution of functional attributes across entity groups to assess functional similarity in a statistically meaningful and biologically interpretable manner. Results on diverse data, including high-throughput and computationally predicted PPIs, as well as structural and computationally inferred DDIs for different organisms show that: (i) the relationship between functional similarity and network proximity is captured in a much more (biologically) intuitive manner by our measure, compared to existing measures and (ii) network proximity and functional similarity are significantly more correlated in DDI networks than in PPI networks, and that structurally determined DDIs provide better functional relevance as compared to computationally inferred DDIs.     

Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness.

Individuals with asthma have increased levels of nitric oxide in their exhaled air. To explore its role, we have developed a regulatable transgenic mouse capable of overexpressing inducible nitric oxide synthase in a lung-specific fashion. The CC10-rtTA-NOS-2 mouse contains two transgenes, a reverse tetracycline transactivator under the control of the Clara cell protein promoter and the mouse nitric oxide synthase-2 (NOS-2) coding region under control of a tetracycline operator. Addition of doxycycline to the drinking water of CC10-rtTA-NOS-2 mice causes an increase in nitric oxide synthase-2 that is largely confined to the airway epithelium. The fraction of expired nitric oxide increases over the first 24 h from approximately 10 parts per billion to a plateau of approximately 20 parts per billion. There were no obvious differences between CC10-rtTA-NOS-2 mice, with or without doxycycline, and wild-type mice in lung histology, bronchoalveolar protein, total cell count, or count differentials. However, airway resistance was lower in CC10-rtTA-NOS-2 mice with doxycycline than in CC10-rtTA-NOS-2 mice without doxycycline or wild-type mice with doxycycline. Moreover, doxycycline-treated CC10-rtTA-NOS-2 mice were hyporesponsive to methacholine compared with other groups. These data suggest that increased nitric oxide in the airways has no proinflammatory effects per se and may have beneficial effects on pulmonary function.     

High-throughput screening of a 100,000-compound library for inhibitors of influenza A virus (H3N2)

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-microM compound concentration against influenza strain A/Udorn/72 (H3N2). The "hit" rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-microM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI(50) = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI(50) > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity.     

Development of new cytoplasmic-genetic male-sterile lines in pigeonpea from crosses betweenCajanus cajan (L. Millsp. andC. scarabaeoides (L.) Thouars

Exploitation of hybrid vigour is quite possible in cross-pollinated crops. However, pigeonpea is a grain legume crop with a moderate level of cross-pollination (20-70%), which is mainly aided by insect pollinators. As a first step, hybrids based on genetic male sterility (GMS) were developed in pigeonpea, but the hybrid seed production technique is not farmer-friendly, because in the hybrid seed production plot 50% of the population, which are male-fertile in the female rows, have to be eliminated in time before contamination. This requires skilled labour and is a time-consuming process, which increases the cost of hybrid seed production. Therefore, the objective of this study was to develop cytoplasmic-genetic male-sterile (CGMS) lines in pigeonpea through wide hybridization, which would be very suitable for hybrid seed production. Two CGMS lines, viz. CORG 990052 A and CORG 990047, were developed by interspecific hybridization of Cajanus cajan and C. scarabaeoides. Restorers were identified and three CGMS-based pigeonpea hybrids were developed. The hybrid COPH 3 is found to be promising in Tamil Nadu State, India.