Dynamics of magnetotactic bacteria in a rotating magnetic field

The dynamics of the motile magnetotactic bacterium Magnetospirillum gryphiswaldense in a rotating magnetic field is investigated experimentally and analyzed by a theoretical model. These elongated bacteria are propelled by single flagella at each bacterial end and contain a magnetic filament formed by a linear assembly of approximately 40 ferromagnetic nanoparticles. The movements of the bacteria in suspension are analyzed by consideration of the orientation of their magnetic dipoles in the field, the hydrodynamic resistance of the bacteria, and the propulsive force of the flagella. Several novel features found in experiments include a velocity reversal during motion in the rotating field and an interesting diffusive wandering of the trajectory curvature centers. A new method to measure the magnetic moment of an individual bacterium is proposed based on the theory developed.     

QSAR of multiple mutated antibodies

The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q(2) = 0.74, R(2) = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.     

Highly efficient production of phosphorylated hepatitis B core particles in yeast Pichia pastoris

Virus-like particles (VLPs) of the recombinant hepatitis B virus (HBV) core protein (HBc) are routinely used in HBV diagnostics worldwide and are of potential interest as carriers of foreign peptides (e.g., immunological epitopes and targeting addresses, and/or as vessels for packaged diagnostic and therapeutic nanomaterials). Despite numerous reports exploiting different expression systems, a rapid and comprehensive large-scale methodology for purification of HBc VLPs from yeast is still lacking. Here, we present a convenient protocol for highly efficient production and rapid purification of endotoxin-free ayw subtype HBc VLPs from the methylotrophic yeast Pichia pastoris. The HBc gene expression cassette along with the geneticin resistance gene was transferred to the P. pastoris genome via homologous recombination. A producer clone was selected among 2000 transformants for the optimal synthesis of the target protein. Fermentation conditions were established ensuring biomass accumulation of 163 g/L. A simple combination of pH/heat and salt treatment followed by a single anion-exchange chromatography step resulted in a more than 90% pure preparation of HBc VLPs, with a yield of about 3.0 mg per 1 g of wet cells. Purification is performed within a day and may be easily scaled up if necessary. The quality of HBc VLPs was verified by electron microscopy. Mass spectrometry analysis and direct polyacrylamide gel staining revealed phosphorylation of HBc at at least two sites. To our knowledge, this is the first report of HBc phosphorylation in yeast.     

Crystal Structure of the Bacteriophage Qβ Coat Protein in Complex with the RNA Operator of the Replicase Gene

The coat proteins of single-stranded RNA bacteriophages specifically recognize and bind to a hairpin structure in their genome at the beginning of the replicase gene. The interaction serves to repress the synthesis of the replicase enzyme late in infection and contributes to the specific encapsidation of phage RNA. While this mechanism is conserved throughout the Leviviridae family, the coat protein and operator sequences from different phages show remarkable variation, serving as prime examples for the co-evolution of protein and RNA structure. To better understand the protein–RNA interactions in this virus family, we have determined the three-dimensional structure of the coat protein from bacteriophage Qβ bound to its cognate translational operator. The RNA binding mode of Qβ coat protein shares several features with that of the widely studied phage MS2, but only one nucleotide base in the hairpin loop makes sequence-specific contacts with the protein. Unlike in other RNA phages, the Qβ coat protein does not utilize an adenine-recognition pocket for binding a bulged adenine base in the hairpin stem but instead uses a stacking interaction with a tyrosine side chain to accommodate the base. The extended loop between β strands E and F of Qβ coat protein makes contacts with the lower part of the RNA stem, explaining the greater length dependence of the RNA helix for optimal binding to the protein. Consequently, the complex structure allows the proposal of a mechanism by which the Qβ coat protein recognizes and discriminates in favor of its cognate RNA.     

Structure and stability of icosahedral particles of a covalent coat protein dimer of bacteriophage MS2

Particles formed by the bacteriophage MS2 coat protein mutants with insertions in their surface loops induce a strong immune response against the inserted epitopes. The covalent dimers created by fusion of two copies of the coat protein gene are more tolerant to various insertions into the surface loops than the single subunits. We determined a 4.7‐Å resolution crystal structure of an icosahedral particle assembled from covalent dimers and compared its stability with wild‐type virions. The structure resembled the wild‐type virion except for the intersubunit linker regions. The covalent dimer orientation was random with respect to both icosahedral twofold and quasi‐twofold symmetry axes. A fraction of the particles was unstable in phosphate buffer because of assembly defects. Our results provide a structural background for design of modified covalent coat protein dimer subunits for use in immunization.     

Investigating the structural basis of purine specificity in the structures of MS2 coat protein RNA translational operator hairpins

We have determined the structures of complexes between the phage MS2 coat protein and variants of the replicase translational operator in order to explore the sequence specificity of the RNA–protein interaction. The 19-nt RNA hairpins studied have substitutions at two positions that have been shown to be important for specific binding. At one of these positions, –10, which is a bulged adenosine (A) in the stem of the wild-type operator hairpin, substitutions were made with guanosine (G), cytidine (C) and two non-native bases, 2-aminopurine (2AP) and inosine (I). At the other position, –7 in the hairpin loop, the native adenine was substituted with a cytidine. Of these, only the G-10, C-10 and C-7 variants showed interpretable density for the RNA hairpin. In spite of large differences in binding affinities, the structures of the variant complexes are very similar to the wild-type operator complex. For G-10 substitutions in hairpin variants that can form bulges at alternative places in the stem, the binding affinity is low and a partly disordered conformation is seen in the electron density maps. The affinity is similar to that of wild-type when the base pairs adjacent to the bulged nucleotide are selected to avoid alternative conformations. Both purines bind in a very similar way in a pocket in the protein. In the C-10 variant, which has very low affinity, the cytidine is partly inserted in the protein pocket rather than intercalated in the RNA stem. Substitution of the wild-type adenosine at position –7 by pyrimidines gives strongly reduced affinities, but the structure of the C-7 complex shows that the base occupies the same position as the A-7 in the wild-type RNA. It is stacked in the RNA and makes no direct contact with the protein.     

Variety of size and form of GRM2 bacterial microcompartment particles

Bacterial microcompartments (BMCs) are bacterial organelles involved in enzymatic processes, such as carbon fixation, choline, ethanolamine and propanediol degradation, and others. Formed of a semi‐permeable protein shell and an enzymatic core, they can enhance enzyme performance and protect the cell from harmful intermediates. With the ability to encapsulate non‐native enzymes, BMCs show high potential for applied use. For this goal, a detailed look into shell form variability is significant to predict shell adaptability. Here we present four novel 3D cryo‐EM maps of recombinant Klebsiella pneumoniae GRM2 BMC shell particles with the resolution in range of 9 to 22 Å and nine novel 2D classes corresponding to discrete BMC shell forms. These structures reveal icosahedral, elongated, oblate, multi‐layered and polyhedral traits of BMCs, indicating considerable variation in size and form as well as adaptability during shell formation processes.     

5-Substituted-(1,2,3-triazol-4-yl)thiophene-2-sulfonamides strongly inhibit human carbonic anhydrases I,II, IX and XII: Solution and X-ray crystallographic studies

We report here a series of 2-thiophene-sulfonamides incorporating 1-substituted aryl-1,2,3-triazolyl moieties, prepared by click chemistry from 5-ethynylthiophene-2-sulfonamide and substituted aryl azides. The new sulfonamides were investigated as inhibitors of the zinc metalloenzyme CA (EC 4.2.1.1), and more specifically against the human (h) cytosolic isoforms hCA I and II and the transmembrane, tumor-associated ones hCA IX and XII: The new compounds were medium–weak hCA I inhibitors (K_Is in the range of 224–7544 nM), but were compactly, highly effective, low nanomolar hCA II inhibitors (K_Is of 2.2–7.7 nM). The tumor-associated hCA IX was inhibited with K_Is ranging between 5.4 and 811 nM, whereas hCA XII with inhibition constants in the range of 3.4–239 nM. The X-ray crystal structure of the adducts of two such compounds bound to hCA II (one incorporating 1-naphthyl, the other one 3-cyanophenyl moieties) evidenced the reasons of the high affinity for hCA II. Highly favorable, predominantly hydrophobic interactions between the sulfonamide scaffold and the hCA II active site were responsible for the binding, in addition to the coordination of the sulfamoyl moiety to the zinc ion. The tails of the two inhibitors adopted very diverse orientations when bound to the active site, with the naphthyltriazolyl moiety orientated towards the hydrophobic half of the active site, and the 3-cyanophenyl one pointing towards the hydrophilic half. These data may be used for the structure-based drug design of even more effective hCA II inhibitors, with potential use as antiglaucoma agents or as diuretics.     

Structure and Function of CutC Choline Lyase from Human Microbiota Bacterium Klebsiella pneumoniae

CutC choline trimethylamine-lyase is an anaerobic bacterial glycyl radical enzyme (GRE) that cleaves choline to produce trimethylamine (TMA) and acetaldehyde. In humans, TMA is produced exclusively by the intestinal microbiota, and its metabolite, trimethylamine oxide, has been associated with a higher risk of cardiovascular diseases. Therefore, information about the three-dimensional structures of TMA-producing enzymes is important for microbiota-targeted drug discovery. We have cloned, expressed, and purified the CutC GRE and the activating enzyme CutD from Klebsiella pneumoniae, a representative of the human microbiota. We have determined the first crystal structures of both the choline-bound and choline-free forms of CutC and have discovered that binding of choline at the ligand-binding site triggers conformational changes in the enzyme structure, a feature that has not been observed for any other characterized GRE.     

Single nucleotide polymorphisms of the purinergic 1 receptor are not associated with myocardial infarction in a Latvian population

The purinergic 1 receptor (P2RY1) has been implicated in development of heart disease and in individual pharmacodynamic response to anticoagulant therapies. However, the association of polymorphisms in the P2RY1 gene with myocardial infarction (MI), and its associated conditions, has yet to be reported in the literature. We evaluated seven known SNPs in P2RY1 for association with MI in a Latvian population. Seven independent parameters that are related to MI [body mass index (BMI), type 2 diabetes (T2D), angina pectoris, hypertension, hyperlipidemia, atrial fibrillation and heart failure] were investigated. No significant association with MI was observed for any of the polymorphisms. Those SNPs for which the P value was close to significance were located in coding or promoter regions. Intriguingly, carriers of the minor allele in the P2RY1 gene locus showed a tendency towards higher onset age for MI, suggesting a possible protective effect of these SNPs against MI or their contribution in progression as opposed to onset. Finally, a linkage disequilibrium (LD) plot was generated for these polymorphisms in the Latvian population. The results of this study suggest that the role of P2RY1 in individuals from Latvian population is likely to be principally involved in platelet aggregation and thromboembolic diseases, and not as a significant contributing factor to the global metabolic syndrome.