CAVER 3.0: A Tool for the Analysis of Transport Pathways in Dynamic Protein Structures

Tunnels and channels facilitate the transport of small molecules, ions and water solvent in a large variety of proteins. Characteristics of individual transport pathways, including their geometry, physico-chemical properties and dynamics are instrumental for understanding of structure-function relationships of these proteins, for the design of new inhibitors and construction of improved biocatalysts. CAVER is a software tool widely used for the identification and characterization of transport pathways in static macromolecular structures. Herein we present a new version of CAVER enabling automatic analysis of tunnels and channels in large ensembles of protein conformations. CAVER 3.0 implements new algorithms for the calculation and clustering of pathways. A trajectory from a molecular dynamics simulation serves as the typical input, while detailed characteristics and summary statistics of the time evolution of individual pathways are provided in the outputs. To illustrate the capabilities of CAVER 3.0, the tool was applied for the analysis of molecular dynamics simulation of the microbial enzyme haloalkane dehalogenase DhaA. CAVER 3.0 safely identified and reliably estimated the importance of all previously published DhaA tunnels, including the tunnels closed in DhaA crystal structures. Obtained results clearly demonstrate that analysis of molecular dynamics simulation is essential for the estimation of pathway characteristics and elucidation of the structural basis of the tunnel gating. CAVER 3.0 paves the way for the study of important biochemical phenomena in the area of molecular transport, molecular recognition and enzymatic catalysis. The software is freely available as a multiplatform command-line application at http://www.caver.cz.

This is a PLOS Computational Biology Software Article

     

Premature centromere division of the X chromosome in neurons in Alzheimer's disease

Premature centromere division (PCD) represents a loss of control over the sequential separation and segregation of chromosome centromeres. Although first described in aging women, PCD on the X chromosome (PCD,X) is markedly elevated in peripheral blood lymphocytes of individuals suffering from Alzheimer disease (AD). The present study evaluated PCD,X, using a fluorescent in situ hybridization method, in interphase nuclei of frontal cerebral cortex neurons from sporadic AD patients and age-matched controls. The average frequency of PCD,X in AD patients (8.60 ± 1.20%) was almost three times higher ( p  < 0.01) than in the control group (2.96 ± 1.20). However, consistent with previous studies, no mitotic cells were found in neurons in either AD or control brain, suggesting an intrinsic inability of post-mitotic neurons to divide. In view of the fact that it has been well-documented that neurons in AD can re-enter into the cell division cycle, the findings presented here of increased PCD advance the hypothesis that deregulation of the cell cycle may contribute to neuronal degeneration and subsequent cognitive deficits in AD.     

Photosynthetic Response of Carrots to Varying Irradiances

Response to irradiance of leaf net photosynthetic rates (P _N) of four carrot cultivars: Cascade, Caro Choice (CC), Oranza, and Red Core Chantenay (RCC) were examined in a controlled environment. Gas exchange measurements were conducted at photosynthetic active radiation (PAR) from 100 to 1 000 μmol m⁻² s⁻¹ at 20 °C and 350 μmol (CO₂) mol⁻¹(air). The values of P _N were fitted to a rectangular hyperbolic nonlinear regression model. P _N for all cultivars increased similarly with increasing PAR but Cascade and Oranza generally had higher P _N than CC. None of the cultivars reached saturation at 1 000 μmol m⁻² s⁻¹. The predicted P _N at saturation (P _Nmax) for Cascade, CC, Oranza, and RCC were 19.78, 16.40, 19.79, and 18.11 μmol (CO₂) m⁻² s⁻¹, respectively. The compensation irradiance (I _c) occurred at 54 μmol m⁻² s⁻¹ for Cascade, 36 μmol m⁻² s⁻¹ for CC, 45 μmol m⁻² s⁻¹ for Oranza, and 25 μmol m⁻² s⁻¹ for RCC. The quantum yield among the cultivars ranged between 0.057–0.033 mol(CO₂) mol⁻¹(PAR) and did not differ. Dark respiration varied from 2.66 μmol m⁻² s⁻¹ for Cascade to 0.85 μmol m⁻² s⁻¹ for RCC. As P _N increased with PAR, intercellular CO₂ decreased in a non-linear manner. Increasing PAR increased stomatal conductance and transpiration rate to a peak between 600 and 800 μmol m⁻² s⁻¹ followed by a steep decline resulting in sharp increases in water use efficiency. This revised version was published online in August 2006 with corrections to the Cover Date.     

Polymorphic nuclear markers for aquatic macroinvertebrates Anisops hackeri, Micronecta gracilis and Necterosoma wollastoni

In Australia, aquatic macroinvertebrates have faced massive changes to their habitats because of human actions. Here, we have developed nuclear, polymorphic markers for the waterboatmen Micronecta gracilis (Hemiptera), a backswimmer Anisops hackeri (Hemiptera), and a diving beetle Necterosoma wollastoni (Coleoptera). These markers will allow us to study gene flow and genetic diversity in these species in massively altered forested and agricultural landscapes.     

Secretion of Recombinant Proteins via the Chaperone/Usher Pathway in Escherichia coli

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1β (hIL-1β), and mature Caf1, the processed product (hIL-1β:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1β:Caf1 in the periplasm. Soluble hIL-1β:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1β. The results indicate that Caf1M-induced release of hIL-1β:Caf1 from the inner membrane promotes folding of the hIL-1β domain. Similar results were obtained with the fusion of Caf1 to hIL-1β receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1β:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1β:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.     

Changes in the expression and the enzymic properties of the 20S proteasome in sugar-starved maize roots. evidence for an in vivo oxidation of the proteasome

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of alpha 3 and beta 6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations.     

Elimination of proton donor strongly affects directionality and efficiency of proton transport in ESR,a light-driven proton pump from Exiguobacterium sibiricum

ESR from Exiguobacterium sibiricum is a retinal protein which functions as a proton pump. Unusual feature of ESR is that a lysine residue is present at a site for the internal proton donor, which in other proton pumps is a carboxylic residue. Replacement of Lys96 with alanine slows reprotonation of the Schiff base by two orders of magnitude, indicating that Lys96 and interacting water molecules function as internal proton donor to the Schiff base. In this work we examined time resolved generation of light-induced electric potential ΔΨ by the K96A mutant reconstituted into proteoliposomes. We found that the ΔΨ component, which accompanied reprotonation of the Schiff base in wild type ESR, was not only slowed but also decreased greatly in the mutant, and negative phase appeared at high pH. This indicates a higher probability of back reactions in ESR than in bacteriorhodopsin since no negative components have been observed in homologous mutants of BR, D96N and D96A. The higher rate of back reactions in ESR is probably caused by different arrangement of the proton acceptor site compared to that in BR and different sequence of proton release and uptake. Addition of sodium azide, which substitutes for the internal proton donor, restores both the rate and amplitude of the ΔΨ components related to the Schiff base reprotonation in the K96A mutant. This indicates that overall proton transport results from competition of forward and reverse reactions, and emphasizes the importance of internal donor for high efficiency and directionality of H⁺ transfer.     

Biogeochemistry of methane and methanogenic archaea in permafrost

This study summarizes the findings of our research on the genesis of methane, its content and distribution in permafrost horizons of different age and origin. Supported by reliable data from a broad geographical sweep, these findings confirm the presence of methane in permanently frozen fine-grained sediments. In contrast to the omnipresence of carbon dioxide in permafrost, methane-containing horizons (up to 40.0 mL kg(-1)) alternate with strata free of methane. Discrete methane-containing horizons representing over tens of thousands of years are indicative of the absence of methane diffusion through the frozen layers. Along with the isotopic composition of CH(4) carbon (delta(13)C -64 per thousand to -99 per thousand), this confirms its biological origin and points to in situ formation of this biogenic gas. Using (14)C-labeled substrates, the possibility of methane formation within permafrost was experimentally shown, as confirmed by delta(13)C values. Extremely low values (near -99 per thousand) indicate that the process of CH(4) formation is accompanied by the substantial fractionation of carbon isotopes. For the first time, cultures of methane-forming archaea, Methanosarcina mazei strain JL01 VKM B-2370, Methanobacterium sp. strain M2 VKM B-2371 and Methanobacterium sp. strain MK4 VKM B-2440 from permafrost, were isolated and described.