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Structural basis of the TAL effector-DNA interaction.
Get PDFPhytopathogen transcription activator-like effectors (TALEs) bind DNA in a sequence specific manner in order to manipulate host transcription. TALE specificity correlates with repeat variable diresidues in otherwise highly stereotypical 34-35mer repeats. Recently, the crystal structures of two TALE DNA-binding domains have illustrated the molecular basis of the TALE cipher. The structures show that the TALE repeats form a right-handed superhelix that is wound around largely undistorted B-DNA to match its helical parameters. Surprisingly, repeat variable residue 1 is not in contact with the bases. Instead, it is involved in hydrogen bonding interactions that stabilize the overall structure of the protein. Repeat variable residue 2 contacts the top strand base and forms sequence-specific hydrogen bonds and/or van der Waals contacts. Very unexpectedly, bottom strand bases are exposed to solvent and do not make any direct contacts with the protein. This review contains a summary of TALE biology and applications and a detailed description of the recent breakthroughs that have provided insights into the molecular basis of the TALE code
RIBER/DIBER: a software suite for crystal content analysis in the studies of protein-nucleic acid complexes
Get PDFSummary: Co-crystallization experiments of proteins with nucleic acids do not guarantee that both components are present in the crystal. We have previously developed DIBER to predict crystal content when protein and DNA are present in the crystallization mix. Here, we present RIBER, which should be used when protein and RNA are in the crystallization drop. The combined RIBER/DIBER suite builds on machine learning techniques to make reliable, quantitative predictions of crystal content for non-expert users and high throughput crystallography.
Availability and Implementation: The program source code, Linux binaries and a webserver are available at http://diber.iimcb.gov.pl/. RIBER/DIBER requires diffraction data to at least 3.0 Å resolution in MTZ or CIF (webserver only) format. The program is written in C/C++ and relies on the CCP4 (Winn, et al., 2011) and Clipper (Cowtan, 2003) libraries for handling diffraction data. The LIBSVM (Chang and Lin, 2011) library is used for decision making. The RIBER/DIBER code is subject to the GNU Public License
Analysis of immunogenicity of different protein groups from malaria parasite Plasmodium falciparum
Get PDFIt was observed that pressure of host immune system leads to diversifying selection (which can be measured in terms of pN/pS ratio). In this research we checked whether Plasmodium falciparum proteins containing experimentally evident epitopes from the IEDB database are subject to diversifying selection. We also investigated which life stage of this parasite and which proteins are subject to the strongest immune pressure. To answer these questions we used information about experimentally evident epitopes from Plasmodium, that interact with human immune system and sequences of different isolates of P. falciparum.
We confirmed the expectations that proteins containing IEDB epitopes are subject to stronger diversifying selection which is evidenced by higher pN/pS ratio. A stage characterized by the highest average pN/pS ratio is that of the sporozoite. The greatest fraction of putative antigens is also present at this stage. We also found that the sporozoite stage is particularly interesting for further analysis as it potentially contains the highest number of unidentified epitopes
Metal and antibiotic resistance of bacteria isolated from the Baltic Sea
Get PDFThe resistance of 49 strains of bacteria isolated from surface Baltic Sea waters to 11 antibiotics was analyzed
and the resistance of selected strains to three metal ions (Ni2+, Mn2+, Zn2+) was tested. Most isolates belonged to
Gammaproteobacteria (78 %), while Alphaproteobacteria (8 %), Actinobacteria (10 %), and Bacteroidetes (4 %) were less
abundant. Compelling evidence for relationships between resistance, and the presence of plasmids and the ability to produce
pigments, as demonstrated in previous reports, was not obtained for the strains isolated in this work. In particular, strains
resistant to multiple antibiotics did not carry plasmids more frequently than sensitive strains. A relation between resistance
and the four aminoglycosides tested (gentamycin, kanamycin, neomycin, and streptomycin), but not to spectinomycin, was
demonstrated. This observation is of interest given that spectinomycin is not always classified as an aminoglycoside because
it lacks a traditional sugar moiety. Statistical analysis indicated relationships with resistance to other antibiotics (ampicillin
and erythromycin, chloramphenicol and erythromycin, chloramphenicol and tetracycline, erythromycin and tetracycline),
suggesting the linkage of resistance genes for antibiotics belonging to different classes. The effects of NiSO4, ZnCl2 and
MnCl2 on various media suggested that the composition of Marine Broth might result in low concentrations of Mn2+ due to
chemical interactions that potentially lead to precipitation
Genomics of staphylococcal Twort-like phages--potential therapeutics of the post-antibiotic era.
No full textPolyvalent bacteriophages of the genus Twort-like that infect clinically relevant Staphylococcus strains may be among the most promising phages with potential therapeutic applications. They are obligatorily lytic, infect the majority of Staphylococcus strains in clinical strain collections, propagate efficiently and do not transfer foreign DNA by transduction. Comparative genomic analysis of 11 S. aureus/S. epidermidis Twort-like phages, as presented in this chapter, emphasizes their strikingly high similarity and clear divergence from phage Twort of the same genus, which might have evolved in hosts of a different species group. Genetically, these phages form a relatively isolated group, which minimizes the risk of acquiring potentially harmful genes. The order of genes in core parts of their 127 to 140-kb genomes is conserved and resembles that found in related representatives of the Spounavirinae subfamily of myoviruses. Functions of certain conserved genes can be predicted based on their homology to prototypical genes of model spounavirus SPO1. Deletions in the genomes of certain phages mark genes that are dispensable for phage development. Nearly half of the genes of these phages have no known homologues. Unique genes are mostly located near termini of the virion DNA molecule and are expressed early in phage development as implied by analysis of their potential transcriptional signals. Thus, many of them are likely to play a role in host takeover. Single genes encode homologues of bacterial virulence-associated proteins. They were apparently acquired by a common ancestor of these phages by horizontal gene transfer but presumably evolved towards gaining functions that increase phage infectivity for bacteria or facilitate mature phage release. Major differences between the genomes of S. aureus/S. epidermidis Twort-like phages consist of single nucleotide polymorphisms and insertions/deletions of short stretches of nucleotides, single genes, or introns of group I. Although the number and location of introns may vary between particular phages, intron shuffling is unlikely to be a major factor responsible for specificity differences
Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal
Get PDFIn this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an
affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction
with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein
in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an
analytical protocol, which provides active protein with a 90% yield and ,100% purity. The method works well in the
presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques
The structural basis for the integrity of adenovirus Ad3 dodecahedron
Get PDFDuring the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical.
Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59 – 61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1 - 47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo
Transgenic tobacco plants as production platform for biologically active human interleukin 2 and its fusion with proteinase inhibitors
Get PDFTransgenic plants offer a low cost approach for the production of pharmaceutically important and commercially valuable recombinant proteins. Our studies were focused on the plant-based production of human interleukin 2 (hIL-2) and its fusion with proteinase inhibitors, either SPI2 from Galleria mellonella or CMTI from Cucurbita maxima. Finally, five plant expression cassettes were obtained. Three of them contained the single cDNA encoding CMTI I, SPI2 and hIL-2, respectively, while two of them contained the translational fusions, SPI2::hIL-2 and CMTI::hIL-2. In all cases, the transgenes were controlled by the RbcS1 promoter and terminator and the recombinant proteins were targeted to the endoplasmic reticulum. After tobacco transformation five groups of transgenic plants were obtained and analyzed. The level of recombinant proteins was estimated either by Western blot or by ELISA.
The biological activity of plant-produced hIL-2 alone or in a fusion with SPI2 or CMTI was confirmed using the mammalian cells proliferation assay. The activities of proteinase inhibitors were confirmed in proteolysis assay using azocoll as a substrate. The usefulness of using proteinase inhibitor CMTI I in a fusion with hIL-2 as a protective agent against trypsin digestion was demonstrated
Structure, regulation and cellular functions of Rab geranylgeranyl transferase and its cellular partner Rab Escort Protein
Get PDFRab geranylgeranyl transferase is an enzyme responsible for double geranylgeranylation of Rab proteins in all eukaryotic cells. In the present article we would like to focus on new findings concerning the holoenzyme structure and mechanism of catalytic activity, its mode of regulation and consequences of RGGT deficiency in different eucaryotic model organisms and patients