On the basis of the crystallographic structures of three nucleic acid intercalation complexes involving ethidium and proflavine, we have analyzed the interaction energies between intercalator chromophores and their four nearest bases, using a hybrid variation-perturbation method at the second-order Møller-Plesset theory level (MP2) with a 6-31G(d,p) basis set. A total MP2 interaction energy minimum precisely reproduces the crystallographic position of the ethidium chromophore in the intercalation plane between UA/AU bases. The electrostatic component constitutes the same fraction of the total energy for all three studied structures. The multipole electrostatic interaction energy, calculated from cumulative atomic multipole moments (CAMMs), was found to converge only after including components above the fifth order. CAMM interaction surfaces, calculated on grids in the intercalation planes of these structures, reasonably reproduce the alignment of intercalators in crystal structures; they exhibit additional minima in the direction of the DNA grooves, however, which also need to be examined at higher theory levels if no crystallographic data are given.
Prior to bacterial cell division, the ATP-dependent polymerization of the cytoskeletal protein, ParA, positions the newly replicated origin-proximal region of the chromosome by interacting with ParB complexes assembled on parS sites located close to the origin. During the formation of unigenomic spores from multi-genomic aerial hyphae compartments of Streptomyces coelicolor, ParA is developmentally triggered to form filaments along the hyphae; this promotes the accurate and synchronized segregation of tens of chromosomes into prespore compartments. Here, we show that in addition to being a segregation protein, ParA also interacts with the polarity protein, Scy, which is a component of the tip-organizing centre that controls tip growth. Scy recruits ParA to the hyphal tips and regulates ParA polymerization. These results are supported by the phenotype of a strain with a mutant form of ParA that uncouples ParA polymerization from Scy. We suggest that the ParA–Scy interaction coordinates the transition from hyphal elongation to sporulation.
Bacteriophages are able to affect the human immune system. Phage-specific antibodies are considered as major factors shaping phage pharmacokinetics and bioavailability. So far, general knowledge of phage antigenicity nevertheless remains extremely limited. Here we present comparative studies of immunogenicity in two therapeutic bacteriophages, A3R and 676Z, active against Staphylococcus aureus, routinely applied in patients at the Phage Therapy Unit, Poland. Comparison of the overall ability of whole phages to induce specific antibodies in a murine model revealed typical kinetics of IgM and IgG induction by these two phages. In further studies we identified the location of four phage proteins in the virions, with the focus on the external capsid head (Mcp) or tail sheath (TmpH) or an unidentified precise location (ORF059 and ORF096), and we confirmed their role as structural proteins of these viruses. Next, we compared the immune response elicited by these proteins after phage administration in mice. Similar to that in T4 phage, Mcp was the major element of the capsid that induced specific antibodies. Studies of protein-specific sera revealed that antibodies specific to ORF096 were able to neutralize antibacterial activity of the phages. In humans (population level), none of the studied proteins plays a particular role in the induction of specific antibodies; thus none potentially affects in a particular way the effectiveness of A3R and 676Z. Also in patients subjected to phage therapy, we did not observe increased specific immune responses to the investigated proteins.
Dynamic
electrostatic catalytic field (DECF) vectors derived from
transition state and reactant wavefunctions for the two-step reaction
occurring within ketosteroid isomerase (KSI) have been calculated
using MP2/aug-cc-pVTZ and lower theory levels to determine the magnitude
of the catalytic effect and the optimal directions of proton transfers
in the KSI hydrogen-bond network. The most surprising and meaningful
finding is that the KSI catalytic activity is enhanced by proton dislocations
proceeding in opposite directions for each of the two consecutive
reaction steps in the same hydrogen network. Such a mechanism allows
an ultrafast switching of the catalytic proton wire environment, possibly
related to the exceptionally high KSI catalytic power.
The A245K mutant of Bacillus stearothermophilus L-lactate dehydrogenase has been expressed in Escherichia coli and purified. A qualitative change in the reaction mechanism prior to the hydride transfer step in the reverse direction in the mutant is revealed. Both transient and steady state characteristics of the mutant are presented and show in contrast to the wild-type enzyme where a rearrangement of an enzyme-NADH-pyruvate complex is rate-limiting that in the mutant the rearrangement is much faster and hydride transfer is the first slow step. The steady state is limited by a new second slower conformation change involving an NAD+ complex. The mutation may provide a valuable framework for inhibitor and drug design research.
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