Tryptophan-free human PNP reveals catalytic site interactions

Human purine nucleoside phosphorylase (PNP) is a homotrimer, containing three nonconserved tryptophan residues at positions 16, 94, and 178, all remote from the catalytic site. The Trp residues were replaced with Tyr to produce Trp-free PNP (Leuko-PNP). Leuko-PNP showed near-normal kinetic properties. It was used (1) to determine the tautomeric form of guanine that produces strong fluorescence when bound to PNP, (2) for thermodynamic binding analysis of binary and ternary complexes with substrates, (3) in temperature-jump perturbation of complexes for evidence of multiple conformational complexes, and (4) to establish the ionization state of a catalytic site tyrosine involved in phosphate nucleophile activation. The (13)C NMR spectrum of guanine bound to Leuko-PNP, its fluorescent properties, and molecular orbital electronic transition analysis establish that its fluorescence originates from the lowest singlet excited state of the N1H, 6-keto, N7H guanine tautomer. Binding of guanine and phosphate to PNP and Leuko-PNP are random, with decreased affinity for formation of ternary complexes. Pre-steady-state kinetics and temperature-jump studies indicate that the ternary complex (enzyme-substrate-phosphate) forms in single binding steps without kinetically significant protein conformational changes as monitored by guanine fluorescence. Spectral changes of Leuko-PNP upon phosphate binding establish that the hydroxyl of Tyr88 is not ionized to the phenolate anion when phosphate is bound. A loop region (residues 243-266) near the purine base becomes highly ordered upon substrate/inhibitor binding. A single Trp residue was introduced into the catalytic loop of Leuko-PNP (Y249W-Leuko-PNP) to determine effects on catalysis and to introduce a fluorescence catalytic site probe. Although Y249W-Leuko-PNP is highly fluorescent and catalytically active, substrate binding did not perturb the fluorescence. Thermodynamic boxes, constructed to characterize the binding of phosphate, guanine, and hypoxanthine to native, Leuko-, and Y249W-Leuko-PNPs, establish that Leuko-PNP provides a versatile protein scaffold for introduction of specific Trp catalytic site probes.     

Electrogenerated chemiluminescence of (bis-bipyridyl)ruthenium(II) acetylacetonate complexes

The synthesis, electrochemistry, spectroscopy and electrogenerated chemiluminescence (ECL) of five bis-bipyridine ruthenium(II) complexes containing acetylacetonate complexes are reported. More specifically, (bpy)₂Ru(BA)₂(PF₆) (bpy = 2,2′-bipyridine; BA = benzoylacetonate), (bpy)₂Ru(TTFA)(PF₆) (TTFA =  thenoyltrifluoroacetonate), (bpy)₂Ru(TFPB)(PF₆) (TFPB = 4,4,4-trifluoro-1-phenyl-1,3-butanedionate), (bpy)₂Ru(TFPD)(PF₆) (TFPD =  1,1,1-trifluoro-2-4-pentanedionate), and (bpy)₂Ru(DBM)(PF₆) (DBM = dibenzoylmethide) display UV-Vis, photoluminescence, electrochemical and ECL properties characteristic of ruthenium bipyridyl complexes. All complexes display absorptions in the UV and visible regions of the spectra, with visible absorptions ranging from 350 to 700 nm, typical of metal-to-ligand charge transfer (MLCT) transitions. Photoluminescence emission maxima are also characteristic of MLCT transitions with wavelength maxima from 575 to 600 nm depending on the nature of the acetylacetonate ligand. ECL efficiencies for the complexes (?_ecl ∼ 0.013-0.051) are much lower than a standard (?_ecl = 1) with electron-withdrawing substituents resulting in lower efficiencies.     

Do Pioneer Cells Exist?

Most mathematical models of collective cell spreading make the standard assumption that the cell diffusivity and cell proliferation rate are constants that do not vary across the cell population. Here we present a combined experimental and mathematical modeling study which aims to investigate how differences in the cell diffusivity and cell proliferation rate amongst a population of cells can impact the collective behavior of the population. We present data from a three-dimensional transwell migration assay that suggests that the cell diffusivity of some groups of cells within the population can be as much as three times higher than the cell diffusivity of other groups of cells within the population. Using this information, we explore the consequences of explicitly representing this variability in a mathematical model of a scratch assay where we treat the total population of cells as two, possibly distinct, subpopulations. Our results show that when we make the standard assumption that all cells within the population behave identically we observe the formation of moving fronts of cells where both subpopulations are well-mixed and indistinguishable. In contrast, when we consider the same system where the two subpopulations are distinct, we observe a very different outcome where the spreading population becomes spatially organized with the more motile subpopulation dominating at the leading edge while the less motile subpopulation is practically absent from the leading edge. These modeling predictions are consistent with previous experimental observations and suggest that standard mathematical approaches, where we treat the cell diffusivity and cell proliferation rate as constants, might not be appropriate.     

Generation of a complement-independent bactericidal IgM against a relapsing fever Borrelia

The spirochetemia of relapsing fever in mice is cleared by a complement-independent, polyclonal IgM response with reactivity to two prominent Ags of 20 and 35 kDa. In this study, we have dissected the polyclonal IgM Ab response against a relapsing fever spirochete to determine the specificity of its complement-independent bactericidal properties. Our experimental approach selectively generated an IgM murine mAb from the early specific immune response to a variable outer membrane protein. This IgM is bactericidal in the absence of complement and is part of the polyclonal Ab response that mediates the clearance of this bacterium from the blood. Purified monoclonal IgM caused direct structural damage to the outer membrane of the spirochete, in the absence of complement, and protected both B cell- and C5-deficient mice from challenge when administered passively. The direct, complement-independent, bactericidal activity of Abs is a critical mechanism of host defense against infection.     

Conformational Itinerary of Pseudomonas aeruginosa 1,6-Anhydro-N-acetylmuramic Acid Kinase during Its Catalytic Cycle

Anhydro-sugar kinases are unique from other sugar kinases in that they must cleave the 1,6-anhydro ring of their sugar substrate to phosphorylate it using ATP. Here we show that the peptidoglycan recycling enzyme 1,6-anhydro-N-acetylmuramic acid kinase (AnmK) from Pseudomonas aeruginosa undergoes large conformational changes during its catalytic cycle, with its two domains rotating apart by up to 32° around two hinge regions to expose an active site cleft into which the substrates 1,6-anhydroMurNAc and ATP can bind. X-ray structures of the open state bound to a nonhydrolyzable ATP analog (AMPPCP) and 1,6-anhydroMurNAc provide detailed insight into a ternary complex that forms preceding an operative Michaelis complex. Structural analysis of the hinge regions demonstrates a role for nucleotide binding and possible cross-talk between the bound ligands to modulate the opening and closing of AnmK. Although AnmK was found to exhibit similar binding affinities for ATP, ADP, and AMPPCP according to fluorescence spectroscopy, small angle x-ray scattering analyses revealed that AnmK adopts an open conformation in solution in the absence of ligand and that it remains in this open state after binding AMPPCP, as we had observed for our crystal structure of this complex. In contrast, the enzyme favored a closed conformation when bound to ADP in solution, consistent with a previous crystal structure of this complex. Together, our findings show that the open conformation of AnmK facilitates binding of both the sugar and nucleotide substrates and that large structural rearrangements must occur upon closure of the enzyme to correctly align the substrates and residues of the enzyme for catalysis.     

Differences in metabolism between the biofilm and planktonic response to metal stress

Bacterial biofilms are known to withstand the effects of toxic metals better than planktonic cultures of the same species. This phenomenon has been attributed to many features of the sessile lifestyle not present in free-swimming populations, but the contribution of intracellular metabolism has not been previously examined. Here, we use a combined GC-MS and (1)H NMR metabolomic approach to quantify whole-cell metabolism in biofilm and planktonic cultures of the multimetal resistant bacterium Pseudomonas fluorescens exposed to copper ions. Metabolic changes in response to metal exposure were found to be significantly different in biofilms compared to planktonic cultures. Planktonic metabolism indicated an oxidative stress response that was characterized by changes to the TCA cycle, glycolysis, pyruvate and nicotinate and niacotinamide metabolism. Similar metabolic changes were not observed in biofilms, which were instead dominated by shifts in exopolysaccharide related metabolism suggesting that metal stress in biofilms induces a protective response rather than the reactive changes observed for the planktonic cells. From these results, we conclude that differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance. An altered metabolic response to metal toxicity represents a novel addition to a growing list of biofilm-specific mechanisms to resist environmental stress.     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

Yeast two hybrid analyses reveal novel binary interactions between human cytomegalovirus-encoded virion proteins

Human cytomegalovirus (HCMV) is the largest human herpesvirus and its virion contains many viral encoded proteins found in the capsid, tegument, and envelope. In this study, we carried out a yeast two-hybrid (YTH) analysis to study potential binary interactions among 56 HCMV-encoded virion proteins. We have tested more than 3,500 pairwise combinations for binary interactions in the YTH analysis, and identified 79 potential interactions that involve 37 proteins. Forty five of the 79 interactions were also identified in human cells expressing the viral proteins by co-immunoprecipitation (co-IP) experiments. To our knowledge, 58 of the 79 interactions revealed by YTH analysis, including those 24 that were also identified in co-IP experiments, have not been reported before. Novel potential interactions were found between viral capsid proteins and tegument proteins, between tegument proteins, between tegument proteins and envelope proteins, and between envelope proteins. Furthermore, both the YTH and co-IP experiments have identified 9, 7, and 5 interactions that were involved with UL25, UL24, and UL89, respectively, suggesting that these "hub" proteins may function as the organizing centers for connecting multiple virion proteins in the mature virion and for recruiting other virion proteins during virion maturation and assembly. Our study provides a framework to study potential interactions between HCMV proteins and investigate the roles of protein-protein interactions in HCMV virion formation or maturation process.