Different forms of African cassava mosaic virus capsid protein within plants and virions

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway

Viscous hydrophilic injection matrices for serial crystallography

Serial (femtosecond) crystallography at synchrotron and X-ray free-electron laser (XFEL) sources distributes the absorbed radiation dose over all crystals used for data collection and therefore allows measurement of radiation damage prone systems, including the use of microcrystals for room-temperature measurements. Serial crystallography relies on fast and efficient exchange of crystals upon X-ray exposure, which can be achieved using a variety of methods, including various injection techniques. The latter vary significantly in their flow rates – gas dynamic virtual nozzle based injectors provide very thin fast-flowing jets, whereas high-viscosity extrusion injectors produce much thicker streams with flow rates two to three orders of magnitude lower. High-viscosity extrusion results in much lower sample consumption, as its sample delivery speed is commensurate both with typical XFEL repetition rates and with data acquisition rates at synchrotron sources. An obvious viscous injection medium is lipidic cubic phase (LCP) as it is used for in meso membrane protein crystallization. However, LCP has limited compatibility with many crystallization conditions. While a few other viscous media have been described in the literature, there is an ongoing need to identify additional injection media for crystal embedding. Critical attributes are reliable injection properties and a broad chemical compatibility to accommodate samples as heterogeneous and sensitive as protein crystals. Here, the use of two novel hydro­gels as viscous injection matrices is described, namely sodium carb­oxy­methyl cellulose and the thermo-reversible block polymer Pluronic F-127. Both are compatible with various crystallization conditions and yield acceptable X-ray background. The stability and velocity of the extruded stream were also analysed and the dependence of the stream velocity on the flow rate was measured. In contrast with previously characterized injection media, both new matrices afford very stable adjustable streams suitable for time-resolved measurements

crystal and solution structures of the multidomain cochaperone DnaJ

Hsp70 chaperones assist in a large variety of protein-folding processes in the cell. Crucial for these activities is the regulation of Hsp70 by Hsp40 cochaperones. DnaJ, the bacterial homologue of Hsp40, stimulates ATP hydrolysis by DnaK (Hsp70) and thus mediates capture of substrate protein, but is also known to possess chaperone activity of its own. The first structure of a complete functional dimeric DnaJ was determined and the mobility of its individual domains in solution was investigated. Crystal structures of the complete molecular cochaperone DnaJ from Thermus thermophilus comprising the J, GF and C-terminal domains and of the J and GF domains alone showed an ordered GF domain interacting with the J domain. Structure-based EPR spin- labelling studies as well as cross-linking results showed the existence of multiple states of DnaJ in solution with different arrangements of the various domains, which has implications for the function of DnaJ.1\. Auflag

Structural Organization of DNA in Chlorella Viruses

Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes

A Novel Mechanism of Programmed Cell Death in Bacteria by Toxin–Antitoxin Systems Corrupts Peptidoglycan Synthesis

Most genomes of bacteria contain toxin–antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections. Finally, we discuss how phosphorylated UNAG likely poisons additional pathways of bacterial cell wall synthesis, making it an attractive lead compound for development of new antibiotics

Hypothesis: Intermediate filament and related proteins: Potential activators of nucleosomes during transcription initiation and elongation?

Intermediate filament (IF) protein tetramers contain two DNA− and core−histone−binding motifs in rotational symmetry in one and the same structural entity. We propose that IF protein oligomers might displace histone octamers from nucleosomes in the process of transcription initiation and elongation, to deposit them transiently on their −helical coiled−coil domains. We further propose that structurally related proteins of the karyoskeleton, constructed from an −helical domain capable of coiled−coil formation and a basic DNA−binding region adjacent to it, may be similarly involved in nucleosome activation. These proteins would function as auxiliary factors that disrupt nucleosomal structure to permit transcription and other DNA−dependent processes to proceed expiditiousl

Gene expression in Acetabularia. I. Calibration of wheat germ cell−free translation system proteins as internal references for two−dimensional electrophoresis

Modification of existing two−dimensional techniques enables isoelectric focusing and sodium dodecyl sulphate polyacrylamide gel electrophoresis of complex mixtures of proteins to be completed within 8 h. The method was optimized to separate the protein components of a wheat germ cell−free translation system, providing a statistically proven resolution better than 0 . 03 of a pH unit for the isoelectric point and 1000 for Mr. Fourteen of the more than 300 proteins separated were characterized with respect to Mr and isoelectric point relative to standard proteins under the same conditions. Stained wheat germ proteins thus serve as internal standards for analysis of in vitro translation product