Publications

TitleAbstractYear(sorted descending)
Filter
PMID
Filter
functional dissection of the multi-domain di-heme cytochrome c(550) from thermus thermophilus.in bacteria, oxidation of sulfite to sulfate, the most common strategy for sulfite detoxification, is mainly accomplished by the molybdenum-containing sulfite:acceptor oxidoreductases (sors). bacterial sors are very diverse proteins; they can exist as monomers or homodimers of their core subunit, as well as heterodimers with an additional cytochrome c subunit. we have previously described the homodimeric sor from thermus thermophilus hb8 (sor(tthb8)), identified its physiological electron accept ...201323383080
mechanism of tetracycline resistance by ribosomal protection protein tet(o).tetracycline resistance protein tet(o), which protects the bacterial ribosome from binding the antibiotic tetracycline, is a translational gtpase with significant similarity in both sequence and structure to the elongation factor ef-g. here, we present an atomic model of the tet(o)-bound 70s ribosome based on our cryo-electron microscopic reconstruction at 9.6-å resolution. this atomic model allowed us to identify the tet(o)-ribosome binding sites, which involve three characteristic loops in dom ...201323403578
crystal structures and molecular dynamics simulations of thermophilic malate dehydrogenase reveal critical loop motion for co-substrate binding.malate dehydrogenase (mdh) catalyzes the conversion of oxaloacetate and malate by using the nad/nadh coenzyme system. the system is used as a conjugate for enzyme immunoassays of a wide variety of compounds, such as illegal drugs, drugs used in therapeutic applications and hormones. we elucidated the biochemical and structural features of mdh from thermus thermophilus (ttmdh) for use in various biotechnological applications. the biochemical characterization of recombinant ttmdh revealed greatly ...201324386145
single molecule analysis of thermus thermophilus ssb protein dynamics on single-stranded dna.single-stranded (ss) dna binding (ssb) proteins play central roles in dna replication, recombination and repair in all organisms. we previously showed that escherichia coli (eco) ssb, a homotetrameric bacterial ssb, undergoes not only rapid ssdna-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssdna. whereas the majority of bacterial ssb family members function as homotetramers, dimeric ssb proteins were recently discovered in a distinct bacter ...201324371279
single molecule analysis of thermus thermophilus ssb protein dynamics on single-stranded dna.single-stranded (ss) dna binding (ssb) proteins play central roles in dna replication, recombination and repair in all organisms. we previously showed that escherichia coli (eco) ssb, a homotetrameric bacterial ssb, undergoes not only rapid ssdna-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssdna. whereas the majority of bacterial ssb family members function as homotetramers, dimeric ssb proteins were recently discovered in a distinct bacter ...201324371279
genetically encoded fluorescent indicator for imaging nad(+)/nadh ratio changes in different cellular compartments.the ratio of nad(+)/nadh is a key indicator that reflects the overall redox state of the cells. until recently, there were no methods for real time nad(+)/nadh monitoring in living cells. genetically encoded fluorescent probes for nad(+)/nadh are fundamentally new approach for studying the nad(+)/nadh dynamics.201324286672
genetically encoded fluorescent indicator for imaging nad(+)/nadh ratio changes in different cellular compartments.the ratio of nad(+)/nadh is a key indicator that reflects the overall redox state of the cells. until recently, there were no methods for real time nad(+)/nadh monitoring in living cells. genetically encoded fluorescent probes for nad(+)/nadh are fundamentally new approach for studying the nad(+)/nadh dynamics.201324286672
structure of escherichia coli rna polymerase holoenzyme at last. 201324272941
directed polymerase evolution.polymerases evolved in nature to synthesize dna and rna, and they underlie the storage and flow of genetic information in all cells. the availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. to circumvent these limit ...201324211837
directed polymerase evolution.polymerases evolved in nature to synthesize dna and rna, and they underlie the storage and flow of genetic information in all cells. the availability of these enzymes for use at the bench has driven a revolution in biotechnology and medicinal research; however, polymerases did not evolve to function efficiently under the conditions required for some applications and their high substrate fidelity precludes their use for most applications that involve modified substrates. to circumvent these limit ...201324211837
identification and characterization of a novel trehalose synthase gene derived from saline-alkali soil metagenomes.a novel trehalose synthase (tres) gene was identified from a metagenomic library of saline-alkali soil by a simple activity-based screening system. sequence analysis revealed that tres encodes a protein of 552 amino acids, with a deduced molecular weight of 63.3 kda. after being overexpressed in escherichia coli and purified, the enzymatic properties of tres were investigated. the recombinant tres displayed its optimal activity at ph 9.0 and 45 °c, and the addition of most common metal ions (1 o ...201324146994
interplay between the trigger loop and the f loop during rna polymerase catalysis.the trigger loop (tl) in the rna polymerase (rnap) active center plays key roles in the reactions of nucleotide addition and rna cleavage catalyzed by rnap. the adjacent f loop (fl) was proposed to contribute to rnap catalysis by modulating structural changes in the tl. here, we investigate the interplay between these two elements during transcription by bacterial rnap. thermodynamic analysis of catalysis by rnap variants with mutations in the tl and fl suggests that the tl is the key element re ...201324089145
interplay between the trigger loop and the f loop during rna polymerase catalysis.the trigger loop (tl) in the rna polymerase (rnap) active center plays key roles in the reactions of nucleotide addition and rna cleavage catalyzed by rnap. the adjacent f loop (fl) was proposed to contribute to rnap catalysis by modulating structural changes in the tl. here, we investigate the interplay between these two elements during transcription by bacterial rnap. thermodynamic analysis of catalysis by rnap variants with mutations in the tl and fl suggests that the tl is the key element re ...201324089145
effects of upconversion nanoparticles on polymerase chain reaction.nanoparticles (nps) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. nucleic acids detection based on upconversion nanoparticles (ucnps), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. we evaluated whether ucnps can improve polymerase chain reactio ...201324039935
exploration of deinococcus-thermus molecular diversity by novel group-specific pcr primers.the deeply branching deinococcus-thermus lineage is recognized as one of the most extremophilic phylum of bacteria. in previous studies, the presence of deinococcus-related bacteria in the hot arid tunisian desert of tataouine was demonstrated through combined molecular and culture-based approaches. similarly, thermus-related bacteria have been detected in tunisian geothermal springs. the present work was conducted to explore the molecular diversity within the deinococcus-thermus phylum in these ...201323996915
computational simulation strategies for analysis of multisubunit rna polymerases. 201323987500
incorporation of nucleoside probes opposite o⁶-methylguanine by sulfolobus solfataricus dna polymerase dpo4: importance of hydrogen bonding.o⁶-methylguanine (o⁶-meg) is a mutagenic dna lesion, arising from the action of methylating agents on guanine (g) in dna. dpo4, an archaeal low-fidelity y-family dna polymerase involved in translesion dna synthesis (tls), is a model for studying how human y-family polymerases bypass dna adducts. previous work showed that dpo4-mediated dttp incorporation is favored opposite o⁶-meg rather than opposite g. however, factors influencing the preference of dpo4 to incorporate dttp opposite o⁶-meg are n ...201323959784
tagetitoxin inhibits transcription by stabilizing pre-translocated state of the elongation complex.transcription elongation consists of repetition of the nucleotide addition cycle: phosphodiester bond formation, translocation and binding of the next nucleotide. inhibitor of multi-subunit rna polymerase tagetitoxin (tgt) enigmatically slows down addition of nucleotides in a sequence-dependent manner, only at certain positions of the template. here, we show that tgt neither affects chemistry of rna synthesis nor induces backward translocation, nor competes with the nucleoside triphosphate (ntp) ...201323935117
energetic and structural details of the trigger-loop closing transition in rna polymerase ii.an evolutionarily conserved element in rna polymerase ii, the trigger loop (tl), has been suggested to play an important role in the elongation rate, fidelity of selection of the matched nucleoside triphosphate (ntp), catalysis of transcription elongation, and translocation in both eukaryotes and prokaryotes. in response to ntp binding, the tl undergoes large conformational changes to switch between distinct open and closed states to tighten the active site and avail catalysis. a computational s ...201323931324
by ribosome possessed. 201323814064
x-ray crystal structures of the escherichia coli rna polymerase in complex with benzoxazinorifamycins.rifampin, a semisynthetic rifamycin, is the cornerstone of current tuberculosis treatment. among many semisynthetic rifamycins, benzoxazinorifamycins have great potential for tb treatment due to their superior affinity for wild-type and rifampin-resistant mycobacterium tuberculosis rna polymerases and their reduced hepatic cyp450 induction activity. in this study, we have determined the crystal structures of the escherichia coli rna polymerase complexes with two benzoxazinorifamycins. the ansa-n ...201323679862
the rna polymerase trigger loop functions in all three phases of the transcription cycle.the trigger loop (tl) forms a conserved element in the rna polymerase active centre that functions in the elongation phase of transcription. here, we show that the tl also functions in transcription initiation and termination. using recombinant variants of rna polymerase from pyrococcus furiosus and a reconstituted transcription system, we demonstrate that the tl is essential for initial rna synthesis until a complete dna-rna hybrid is formed. the archaeal tl is further important for transcripti ...201323737452
a structural role for the php domain in e. coli dna polymerase iii.in addition to the core catalytic machinery, bacterial replicative dna polymerases contain a polymerase and histidinol phosphatase (php) domain whose function is not entirely understood. the php domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. in e. coli dna polymerase iii, however, the php domain has lost several metal-coordinating residues and is likely to be catalytically inactive.201323672456
a rex family transcriptional repressor influences h2o2 accumulation by enterococcus faecalis.rex factors are bacterial transcription factors thought to respond to the cellular nad(+)/nadh ratio in order to modulate gene expression by differentially binding dna. to date, rex factors have been implicated in regulating genes of central metabolism, oxidative stress response, and biofilm formation. the genome of enterococcus faecalis, a low-gc gram-positive opportunistic pathogen, encodes ef2638, a putative rex factor. to study the role of e. faecalis rex, we purified ef2638 and evaluated it ...201323417491
thermostable mismatch-recognizing protein muts suppresses nonspecific amplification during polymerase chain reaction (pcr).polymerase chain reaction (pcr)-related technologies are hampered mainly by two types of error: nonspecific amplification and dna polymerase-generated mutations. here, we report that both errors can be suppressed by the addition of a dna mismatch-recognizing protein, muts, from a thermophilic bacterium. although it had been expected that muts has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. on the basis of this find ...201323519109
structure of the poliiiα-τc-dna complex suggests an atomic model of the replisome.the c-terminal domain (ctd) of the τ subunit of the clamp loader (τc) binds to both the dnab helicase and the dna polymerase iii α subunit (poliiiα), and determines their relative positions and orientations on the leading and lagging strands. here, we present a 3.2 å resolution structure of thermus aquaticus poliiiα in complex with τc and a dna substrate. the structure reveals that the ctd of τc interacts with the ctd of poliiiα through its c-terminal helix and the adjacent loop. additionally, i ...201323478062
x-ray crystal structure of escherichia coli rna polymerase σ70 holoenzyme.escherichia coli rna polymerase (rnap) is the most studied bacterial rnap and has been used as the model rnap for screening and evaluating potential rnap-targeting antibiotics. however, the x-ray crystal structure of e. coli rnap has been limited to individual domains. here, i report the x-ray structure of the e. coli rnap σ(70) holoenzyme, which shows σ region 1.1 (σ1.1) and the α subunit c-terminal domain for the first time in the context of an intact rnap. σ1.1 is positioned at the rnap dna-b ...201323389035
structural basis of transcriptional pausing in bacteria.transcriptional pausing by multisubunit rna polymerases (rnaps) is a key mechanism for regulating gene expression in both prokaryotes and eukaryotes and is a prerequisite for transcription termination. pausing and termination states are thought to arise through a common, elemental pause state that is inhibitory for nucleotide addition. we report three crystal structures of thermus rnap elemental paused elongation complexes (epecs). the structures reveal the same relaxed, open-clamp rnap conforma ...201323374340
whole genome sequencing of thermus oshimai jl-2 and thermus thermophilus jl-18, incomplete denitrifiers from the united states great basin.the strains thermus oshimai jl-2 and thermus thermophilus jl-18 each have a circular chromosome, 2.07 mb and 1.9 mb in size, respectively, and each has two plasmids ranging from 0.27 mb to 57.2 kb. the megaplasmid of each strain contains a gene cluster for the reduction of nitrate to nitrous oxide, consistent with their incomplete denitrification phenotypes.201323405355
redox specificity of 2-hydroxyacid-coupled nad(+)/nadh dehydrogenases: a study exploiting "reactive" arginine as a reporter of protein electrostatics.with "reactive" arginine as a kinetic reporter, 2-hydroxyacid dehydrogenases are assessed in basis of their specialization as nad(+)-reducing or nadh-oxidizing enzymes. specifically, m4 and h4 lactate dehydrogenases (ldhs) and cytoplasmic and mitochondrial malate dehydrogenases (mdhs) are compared to assess if their coenzyme specificity may involve electrostatics of cationic or neutral nicotinamide structure as the basis. the enzymes from diverse eukaryote and prokaryote sources thus are assesse ...201324391777
misfolding of amyloidogenic proteins and their interactions with membranes.in this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. many of these proteins are implicated in serious illnesses such as alzheimer's disease and parkinson's disease. misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membran ...201324970204
misfolding of amyloidogenic proteins and their interactions with membranes.in this paper, we discuss amyloidogenic proteins, their misfolding, resulting structures, and interactions with membranes, which lead to membrane damage and subsequent cell death. many of these proteins are implicated in serious illnesses such as alzheimer's disease and parkinson's disease. misfolding of amyloidogenic proteins leads to the formation of polymorphic oligomers and fibrils. oligomeric aggregates are widely thought to be the toxic species, however, fibrils also play a role in membran ...201324970204
allosteric regulation in phosphofructokinase from the extreme thermophile thermus thermophilus.an investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (pfk) from the extreme thermophile thermus thermophilus (ttpfk) reveals an enzyme that is inhibited by pep and activated by adp by modifying the affinity exhibited for the substrate fructose 6-phosphate (fru-6-p) in a manner analogous to other prokaryotic pfks. however, ttpfk binds both of these allosteric ligands significantly more tightly than other bacterial pfks while effecting a substantially more ...201324328040
allosteric regulation in phosphofructokinase from the extreme thermophile thermus thermophilus.an investigation into the kinetics and regulatory properties of the type-1 phosphofructokinase (pfk) from the extreme thermophile thermus thermophilus (ttpfk) reveals an enzyme that is inhibited by pep and activated by adp by modifying the affinity exhibited for the substrate fructose 6-phosphate (fru-6-p) in a manner analogous to other prokaryotic pfks. however, ttpfk binds both of these allosteric ligands significantly more tightly than other bacterial pfks while effecting a substantially more ...201324328040
structure-based cleavage mechanism of thermus thermophilus argonaute dna guide strand-mediated dna target cleavage.we report on crystal structures of ternary thermus thermophilus argonaute (ttago) complexes with 5'-phosphorylated guide dna and a series of dna targets. these ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of mg(2+) cations, and the putative water nucleophile positioned for in-line attack on the cleavable ph ...201324374628
structure-based cleavage mechanism of thermus thermophilus argonaute dna guide strand-mediated dna target cleavage.we report on crystal structures of ternary thermus thermophilus argonaute (ttago) complexes with 5'-phosphorylated guide dna and a series of dna targets. these ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of mg(2+) cations, and the putative water nucleophile positioned for in-line attack on the cleavable ph ...201324374628
mutant lv(476-7)aa of a-subunit of enterococcus hirae v1-atpase: high affinity of a3b3 complex to df axis and low atpase activity.vacuolar atpase (v-atpase) of enterococcus hirae is composed of a soluble functional domain v1 (a3b3df) and an integral membrane domain vo (ac), where v1 and vo domains are connected by a central stalk, composed of d-, f-, and d-subunits; and two peripheral stalks (e- and g-subunits). we identified 120 interacting residues of a3b3 heterohexamer with d-subunit in df heterodimer in the crystal structures of a3b3 and a3b3df. in our previous study, we reported 10 mutants of e. hirae v1-atpase, which ...201324404436
distinct trna recognition strategies used by a homologous family of editing domains prevent mistranslation.errors in protein synthesis due to mispairing of amino acids with trnas jeopardize cell viability. several checkpoints to prevent formation of ala- and cys-trna(pro) have been described, including the ala-specific editing domain (ins) of most bacterial prolyl-trna synthetases (prorss) and an autonomous single-domain ins homolog, ybak, which clears cys-trna(pro) in trans. in many species where prors lacks an ins domain, proxp-ala, another single-domain ins-like protein, is responsible for editing ...201324371276
distinct trna recognition strategies used by a homologous family of editing domains prevent mistranslation.errors in protein synthesis due to mispairing of amino acids with trnas jeopardize cell viability. several checkpoints to prevent formation of ala- and cys-trna(pro) have been described, including the ala-specific editing domain (ins) of most bacterial prolyl-trna synthetases (prorss) and an autonomous single-domain ins homolog, ybak, which clears cys-trna(pro) in trans. in many species where prors lacks an ins domain, proxp-ala, another single-domain ins-like protein, is responsible for editing ...201324371276
active site plasticity enables metal-dependent tuning of cas5d nuclease activity in crispr-cas type i-c system.clustered regularly interspaced short palindromic repeat (crispr) in association with crispr-associated (cas) proteins constitutes a formidable defense system against mobile genetic elements in prokaryotes. in type i-c, the ribonucleoprotein surveillance complex comprises only three cas proteins, namely, cas5d, csd1 and csd2. unlike type i-e that uses cse3/case for metal-independent crispr rna maturation, type i-c that lacks this deputes cas5d to process the pre-crrna. here, we report the promis ...201324371266
active site plasticity enables metal-dependent tuning of cas5d nuclease activity in crispr-cas type i-c system.clustered regularly interspaced short palindromic repeat (crispr) in association with crispr-associated (cas) proteins constitutes a formidable defense system against mobile genetic elements in prokaryotes. in type i-c, the ribonucleoprotein surveillance complex comprises only three cas proteins, namely, cas5d, csd1 and csd2. unlike type i-e that uses cse3/case for metal-independent crispr rna maturation, type i-c that lacks this deputes cas5d to process the pre-crrna. here, we report the promis ...201324371266
measurement of the intrinsic variability within protein crystals: implications for sample-evaluation and data-collection strategies.the advent of micro-focused x-ray beams has led to the development of a number of advanced methods of sample evaluation and data collection. in particular, multiple-position data-collection and helical oscillation strategies are now becoming commonplace in order to alleviate the problems associated with radiation damage. however, intra-crystal and inter-crystal variation means that it is not always obvious on which crystals or on which region or regions of a crystal these protocols should be per ...201324419635
measurement of the intrinsic variability within protein crystals: implications for sample-evaluation and data-collection strategies.the advent of micro-focused x-ray beams has led to the development of a number of advanced methods of sample evaluation and data collection. in particular, multiple-position data-collection and helical oscillation strategies are now becoming commonplace in order to alleviate the problems associated with radiation damage. however, intra-crystal and inter-crystal variation means that it is not always obvious on which crystals or on which region or regions of a crystal these protocols should be per ...201324419635
lysine carboxylation: unveiling a spontaneous post-translational modification.the carboxylation of lysine residues is a post-translational modification (ptm) that plays a critical role in the catalytic mechanisms of several important enzymes. it occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. its full impact is unknown. in this work, the signature microenvironment of lysine-carboxylation sites has been characterized. in addition, a computational method called predictor of lysine carboxylation (prelyscar) for the de ...201324419378
lysine carboxylation: unveiling a spontaneous post-translational modification.the carboxylation of lysine residues is a post-translational modification (ptm) that plays a critical role in the catalytic mechanisms of several important enzymes. it occurs spontaneously under certain physicochemical conditions, but is difficult to detect experimentally. its full impact is unknown. in this work, the signature microenvironment of lysine-carboxylation sites has been characterized. in addition, a computational method called predictor of lysine carboxylation (prelyscar) for the de ...201324419378
asymmetric chromosome segregation in xanthomonas citri ssp. citri.this study was intended to characterize the chromosome segregation process of xanthomonas citri ssp. citri (xac) by investigating the functionality of the parb factor encoded on its chromosome, and its requirement for cell viability and virulence. using tap tagging we show that parb is expressed in xac. disruption of parb increased the cell doubling time and precluded the ability of xac to colonize the host citrus. moreover, xac mutant cells expressing only truncated forms of parb exhibited the ...201324339434
asymmetric chromosome segregation in xanthomonas citri ssp. citri.this study was intended to characterize the chromosome segregation process of xanthomonas citri ssp. citri (xac) by investigating the functionality of the parb factor encoded on its chromosome, and its requirement for cell viability and virulence. using tap tagging we show that parb is expressed in xac. disruption of parb increased the cell doubling time and precluded the ability of xac to colonize the host citrus. moreover, xac mutant cells expressing only truncated forms of parb exhibited the ...201324339434
functional domains of the 50s subunit mature late in the assembly process.despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. here, we analyze the late stages of assembly of the 50s subunit using bacillus subtilis cells depleted of rbga, a highly conserved gtpase. we found that rbga-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins l16, l27, l28, l33a, l35 and l36. using a novel pulse labeling ...201324335279
functional domains of the 50s subunit mature late in the assembly process.despite the identification of many factors that facilitate ribosome assembly, the molecular mechanisms by which they drive ribosome biogenesis are poorly understood. here, we analyze the late stages of assembly of the 50s subunit using bacillus subtilis cells depleted of rbga, a highly conserved gtpase. we found that rbga-depleted cells accumulate late assembly intermediates bearing sub-stoichiometric quantities of ribosomal proteins l16, l27, l28, l33a, l35 and l36. using a novel pulse labeling ...201324335279
mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from enterococcus faecalis.ef1143 from enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dntp) triphosphohydrolase (dntpase), converting dntps into the deoxyribonucleosides and triphosphate. the dntpase activity of ef1143 is regulated by canonical dntps, which simultaneously act as substrates and activity modulators. previous crystal structures of apo-ef1143 and the protein bound to both dgtp and datp suggested allosteric regu ...201324338016
mechanisms of allosteric activation and inhibition of the deoxyribonucleoside triphosphate triphosphohydrolase from enterococcus faecalis.ef1143 from enterococcus faecalis, a life-threatening pathogen that is resistant to common antibiotics, is a homo-tetrameric deoxyribonucleoside triphosphate (dntp) triphosphohydrolase (dntpase), converting dntps into the deoxyribonucleosides and triphosphate. the dntpase activity of ef1143 is regulated by canonical dntps, which simultaneously act as substrates and activity modulators. previous crystal structures of apo-ef1143 and the protein bound to both dgtp and datp suggested allosteric regu ...201324338016
identification of a selective polymerase enables detection of n(6)-methyladenosine in rna.n(6)-methyladenosine (m(6)a) is the most abundant mrna modification and has important links to human health. while recent studies have successfully identified thousands of mammalian rna transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m(6)a. here we have identified a polymerase with reverse transcriptase activity (from thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified a over ...201324328136
measuring the shapes of macromolecules - and why it matters.the molecular basis of life rests on the activity of biological macromolecules, mostly nucleic acids and proteins. a perhaps surprising finding that crystallized over the last handful of decades is that geometric reasoning plays a major role in our attempt to understand these activities. in this paper, we address this connection between geometry and biology, focusing on methods for measuring and characterizing the shapes of macromolecules. we briefly review existing numerical and analytical appr ...201324688748
identification of residues required for stalled-ribosome rescue in the codon-independent release factor yaej.the yaej protein is a codon-independent release factor with peptidyl-trna hydrolysis (pth) activity, and functions as a stalled-ribosome rescue factor in escherichia coli. to identify residues required for yaej function, we performed mutational analysis for in vitro pth activity towards rescue of ribosomes stalled on a non-stop mrna, and for ribosome-binding efficiency. we focused on residues conserved among bacterial yaej proteins. additionally, we determined the solution structure of the ggq d ...201324322300
identification of residues required for stalled-ribosome rescue in the codon-independent release factor yaej.the yaej protein is a codon-independent release factor with peptidyl-trna hydrolysis (pth) activity, and functions as a stalled-ribosome rescue factor in escherichia coli. to identify residues required for yaej function, we performed mutational analysis for in vitro pth activity towards rescue of ribosomes stalled on a non-stop mrna, and for ribosome-binding efficiency. we focused on residues conserved among bacterial yaej proteins. additionally, we determined the solution structure of the ggq d ...201324322300
the mononuclear metal center of type-i dihydroorotase from aquifex aeolicus.dihydroorotase (dho) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. e. coli dho was initially thought to have a mononuclear metal center, but the subsequent x-ray structure clearly showed two zinc ions, α and β, at the catalytic site. aquifex aeolicus dho, is a dodecamer comprised of six dho and six aspartate transcarbamoylase (atc) subunits. the isolated dho monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, ...201324314009
structure of the ribosome with elongation factor g trapped in the pretranslocation state.during protein synthesis, trnas and their associated mrna codons move sequentially on the ribosome from the a (aminoacyl) site to the p (peptidyl) site to the e (exit) site in a process catalyzed by a universally conserved ribosome-dependent gtpase [elongation factor g (ef-g) in prokaryotes and elongation factor 2 (ef-2) in eukaryotes]. although the high-resolution structure of ef-g bound to the posttranslocation ribosome has been determined, the pretranslocation conformation of the ribosome bou ...201324324137
posttranscriptional rna modifications: playing metabolic games in a cell's chemical legoland.nature combines existing biochemical building blocks, at times with subtlety of purpose. rna modifications are a prime example of this, where standard rna nucleosides are decorated with chemical groups and building blocks that we recall from our basic biochemistry lectures. the result: a wealth of chemical diversity whose full biological relevance has remained elusive despite being public knowledge for some time. here, we highlight several modifications that, because of their chemical intricacy, ...201324315934
posttranscriptional rna modifications: playing metabolic games in a cell's chemical legoland.nature combines existing biochemical building blocks, at times with subtlety of purpose. rna modifications are a prime example of this, where standard rna nucleosides are decorated with chemical groups and building blocks that we recall from our basic biochemistry lectures. the result: a wealth of chemical diversity whose full biological relevance has remained elusive despite being public knowledge for some time. here, we highlight several modifications that, because of their chemical intricacy, ...201324315934
structure-function features of a mycoplasma glycolipid synthase derived from structural data integration, molecular simulations, and mutational analysis.glycoglycerolipids are structural components of mycoplasma membranes with a fundamental role in membrane properties and stability. their biosynthesis is mediated by glycosyltransferases (gt) that catalyze the transfer of glycosyl units from a sugar nucleotide donor to diacylglycerol. the essential function of glycolipid synthases in mycoplasma viability, and the absence of glycoglycerolipids in animal host cells make these gt enzymes a target for drug discovery by designing specific inhibitors. ...201324312618
flexibility within the rotor and stators of the vacuolar h+-atpase.the v-atpase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an atp-driven 3-stroke rotary motor (v1) to a multistroke proton pump (vo). this is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. we have used electron microscop ...201324312643
visualization of the joining of ribosomal subunits reveals the presence of 80s ribosomes in the nucleus.in eukaryotes the 40s and 60s ribosomal subunits are assembled in the nucleolus, but there appear to be mechanisms preventing mrna binding, 80s formation, and initiation of translation in the nucleus. to visualize association between ribosomal subunits, we tagged pairs of drosophila ribosomal proteins (rps) located in different subunits with mutually complementing halves of fluorescent proteins. pairs of tagged rps expected to interact, or be adjacent in the 80s structure, showed strong fluoresc ...201324129492
the central role of protein s12 in organizing the structure of the decoding site of the ribosome.the ribosome decodes mrna by monitoring the geometry of codon-anticodon base-pairing using a set of universally conserved 16s rrna nucleotides within the conformationally dynamic decoding site. by applying single-molecule fret and x-ray crystallography, we have determined that conditional-lethal, streptomycin-dependence mutations in ribosomal protein s12 interfere with trna selection by allowing conformational distortions of the decoding site that impair gtpase activation of ef-tu during the trn ...201324152548
intra- and intermolecular regulatory interactions in upf1, the rna helicase central to nonsense-mediated mrna decay in yeast.rna helicases are involved in almost every aspect of rna metabolism, yet very little is known about the regulation of this class of enzymes. in saccharomyces cerevisiae, the stability and translational fidelity of nonsense-containing mrnas are controlled by the group i rna helicase upf1 and the proteins it interacts with, upf2 and upf3. combining the yeast two-hybrid system with genetic analysis, we show here that the cysteine- and histidine-rich (ch) domain and the rna helicase domain of yeast ...201324100012
membrane chaperone secdf plays a role in the secretion of listeria monocytogenes major virulence factors.listeria monocytogenes is a gram-positive human intracellular pathogen that infects diverse mammalian cells. upon invasion, l. monocytogenes secretes multiple virulence factors that target host cellular processes and promote infection. it has been presumed, but was not empirically established, that the sec translocation system is the primary mediator of this secretion. here, we validate an important role for secdf, a component of the sec system, in the secretion of several critical l. monocytoge ...201324056100
functional role of methylation of g518 of the 16s rrna 530 loop by gidb in mycobacterium tuberculosis.posttranscriptional modifications of bacterial rrna serve a variety of purposes, from stabilizing ribosome structure to preserving its functional integrity. here, we investigated the functional role of one rrna modification in particular-the methylation of guanosine at position 518 (g518) of the 16s rrna in mycobacterium tuberculosis. based on previously reported evidence that g518 is located 5 å; from proline 44 of ribosomal protein s12, which interacts directly with the mrna wobble position of ...201324100503
tools for characterizing bacterial protein synthesis inhibitors.many antibiotics inhibit the growth of sensitive bacteria by interfering with ribosome function. however, discovery of new protein synthesis inhibitors is curbed by the lack of facile techniques capable of readily identifying antibiotic target sites and modes of action. furthermore, the frequent rediscovery of known antibiotic scaffolds, especially in natural product extracts, is time-consuming and expensive and diverts resources that could be used toward the isolation of novel lead molecules. i ...201324041905
cloning, expression, purification, crystallization and preliminary x-ray studies of argininosuccinate lyase (rv1659) from mycobacterium tuberculosis.the last enzyme in the arginine-biosynthesis pathway, argininosuccinate lyase, from mycobacterium tuberculosis has been cloned, expressed, purified and crystallized, and preliminary x-ray studies have been carried out on the crystals. the his-tagged tetrameric enzyme with a subunit molecular weight of 50.9 kda crystallized with two tetramers in the asymmetric unit of the orthorhombic unit cell, space group p2(1)2(1)2(1). molecular-replacement calculations and self-rotation calculations confirmed ...201324316845
dna binding properties of human cdc45 suggest a function as molecular wedge for dna unwinding.the cell division cycle protein 45 (cdc45) represents an essential replication factor that, together with the mcm2-7 complex and the four subunits of gins, forms the replicative dna helicase in eukaryotes. recombinant human cdc45 (hcdc45) was structurally characterized and its dna-binding properties were determined. synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle x-ray scattering and atomic force microscopy revealed that hcdc45 exists as an alpha-heli ...201324293646
dna binding properties of human cdc45 suggest a function as molecular wedge for dna unwinding.the cell division cycle protein 45 (cdc45) represents an essential replication factor that, together with the mcm2-7 complex and the four subunits of gins, forms the replicative dna helicase in eukaryotes. recombinant human cdc45 (hcdc45) was structurally characterized and its dna-binding properties were determined. synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle x-ray scattering and atomic force microscopy revealed that hcdc45 exists as an alpha-heli ...201324293646
purification, crystallization and preliminary x-ray studies of mbtn (rv1346) from mycobacterium tuberculosis.in mycobacterium tuberculosis, the protein mbtn (rv1346) catalyzes the formation of a double bond in the fatty-acyl moiety of the siderophore mycobactin, which is used by this organism to acquire essential iron. mbtn is homologous to acyl-coa dehydrogenases, whose general role is to catalyze the α,β-dehydrogenation of fatty-acyl-coa conjugates. mycobactins, however, contain a long unsaturated fatty-acid chain with an unusual cis double bond conjugated to the carbonyl group of the mycobactin core ...201324316828
conserved distal loop residues in the hsp104 and clpb middle domain contact nucleotide-binding domain 2 and enable hsp70-dependent protein disaggregation.the homologous hexameric aaa(+) proteins, hsp104 from yeast and clpb from bacteria, collaborate with hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. how hsp104 and clpb coordinate polypeptide handover with hsp70 is not understood. here, we define conserved distal loop residues between middle domain (md) helix 1 and 2 that are unexpectedly critical for hsp104 and clpb collaboration with hsp70. surprisingly, the hsp104 and clpb md dista ...201324280225
conserved distal loop residues in the hsp104 and clpb middle domain contact nucleotide-binding domain 2 and enable hsp70-dependent protein disaggregation.the homologous hexameric aaa(+) proteins, hsp104 from yeast and clpb from bacteria, collaborate with hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. how hsp104 and clpb coordinate polypeptide handover with hsp70 is not understood. here, we define conserved distal loop residues between middle domain (md) helix 1 and 2 that are unexpectedly critical for hsp104 and clpb collaboration with hsp70. surprisingly, the hsp104 and clpb md dista ...201324280225
structural and physiological analyses of the alkanesulphonate-binding protein (ssua) of the citrus pathogen xanthomonas citri.the uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the xanthomonas genus, which encompasses several pathogenic species. in the present study, we characterised the alkanesulphonate uptak ...201324282519
redox regulation of the calvin-benson cycle: something old, something new.reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, s-nitrosylation, and s-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. these modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. early studies in photosynthetic organisms have identified the calvin-benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated proces ...201324324475
structure of the proteus vulgaris higb-(higa)2-higb toxin-antitoxin complex.bacterial toxin-antitoxin (ta) systems regulate key cellular processes to promote cell survival during periods of stress. during steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. we solved two x-ray crystal structures of the proteus vulgaris tetrameric higb-(higa)2-higb ta complex and found that, unlike most other ta systems, the antitoxin higa makes minimal interactions with toxin higb. higb adop ...201324257752
structure of the proteus vulgaris higb-(higa)2-higb toxin-antitoxin complex.bacterial toxin-antitoxin (ta) systems regulate key cellular processes to promote cell survival during periods of stress. during steady-state cell growth, antitoxins typically interact with their cognate toxins to inhibit activity presumably by preventing substrate recognition. we solved two x-ray crystal structures of the proteus vulgaris tetrameric higb-(higa)2-higb ta complex and found that, unlike most other ta systems, the antitoxin higa makes minimal interactions with toxin higb. higb adop ...201324257752
crystal structure of the γ-hydroxymuconic semialdehyde dehydrogenase from pseudomonas sp. strainwbc-3, a key enzyme involved in para-nitrophenol degradation.para-nitrophenol (pnp) is a highly toxic compound with threats to mammalian health. the pnpe-encoded γ-hydroxymuconic semialdehyde dehydrogenase catalyzes the reduction of γ-hydroxymuconic semialdehyde to maleylacetate in pseudomonas sp. strain wbc-3, playing a key role in the catabolism of pnp to krebs cycle intermediates. however, the catalyzing mechanism by pnpe has not been well understood.201324252642
dynamic dna underpins chromosome dynamics.a recent article by meyer and co-workers provides a detailed description of the dynamics of base-step conformational transitions and of the first steps to the basepair opening. these results underpin the essential role that dna dynamics place in the dna structural transitions that accompany the active processes of transcription, replication, and recombination in bacterial and eukaryotic chromatin.201324268134
molecular basis of adp inhibition of vacuolar (v)-type atpase/synthase.reduction of atp hydrolysis activity of vacuolar-type atpase/synthase (v0v1) as a result of adp inhibition occurs as part of the normal mechanism of v0v1 of thermus thermophilus but not v0v1 of enterococcus hirae or eukaryotes. to investigate the molecular basis for this difference, domain-swapped chimeric v1 consisting of both t. thermophilus and e. hirae enzymes were generated, and their function was analyzed. the data showed that the interaction between the nucleotide binding and c-terminal d ...201324247239
molecular basis of adp inhibition of vacuolar (v)-type atpase/synthase.reduction of atp hydrolysis activity of vacuolar-type atpase/synthase (v0v1) as a result of adp inhibition occurs as part of the normal mechanism of v0v1 of thermus thermophilus but not v0v1 of enterococcus hirae or eukaryotes. to investigate the molecular basis for this difference, domain-swapped chimeric v1 consisting of both t. thermophilus and e. hirae enzymes were generated, and their function was analyzed. the data showed that the interaction between the nucleotide binding and c-terminal d ...201324247239
analysis of the mechanism of nucleosome survival during transcription.maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. our previous data identified a key intermediate (a small intranucleosomal dna loop, ø-loop) that is likely required for nucleosome survival during transcription by rna polymerase ii (pol ii) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. to evaluate these predictions, we analysed transcription through ...201324234452
analysis of the mechanism of nucleosome survival during transcription.maintenance of nucleosomal structure in the cell nuclei is essential for cell viability, regulation of gene expression and normal aging. our previous data identified a key intermediate (a small intranucleosomal dna loop, ø-loop) that is likely required for nucleosome survival during transcription by rna polymerase ii (pol ii) through chromatin, and suggested that strong nucleosomal pausing guarantees efficient nucleosome survival. to evaluate these predictions, we analysed transcription through ...201324234452
comparative xylose metabolism among the ascomycetes c. albicans, s. stipitis and s. cerevisiae.the ascomycetes candida albicans, saccharomyces cerevisiae and scheffersomyces stipitis metabolize the pentose sugar xylose very differently. s. cerevisiae fails to grow on xylose, while c. albicans can grow, and s. stipitis can both grow and ferment xylose to ethanol. however, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. we have create ...201324236198
overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.the psychrophilic, hydrocarbonoclastic microorganism colwellia psychrerythraea is important in global nutrient cycling and bioremediation. in order to investigate how this organism can live so efficiently at low temperatures (~4°c), thermal denaturation studies of a small electron transfer protein from colwellia were performed. colwellia cytochrome c552 was overexpressed in escherichia coli, isolated, purified, and characterized by uv-visible absorption spectroscopy. the melting temperature (tm) ...201324275750
overexpression, purification, and enthalpy of unfolding of ferricytochrome c552 from a psychrophilic microorganism.the psychrophilic, hydrocarbonoclastic microorganism colwellia psychrerythraea is important in global nutrient cycling and bioremediation. in order to investigate how this organism can live so efficiently at low temperatures (~4°c), thermal denaturation studies of a small electron transfer protein from colwellia were performed. colwellia cytochrome c552 was overexpressed in escherichia coli, isolated, purified, and characterized by uv-visible absorption spectroscopy. the melting temperature (tm) ...201324275750
biosynthesis of rare hexoses using microorganisms and related enzymes.rare sugars, referred to as monosaccharides and their derivatives that rarely exist in nature, can be applied in many areas ranging from foodstuffs to pharmaceutical and nutrition industry, or as starting materials for various natural products and drug candidates. unfortunately, an important factor restricting the utilization of rare sugars is their limited availability, resulting from limited synthetic methods. nowadays, microbial and enzymatic transformations have become a very powerful tool i ...201324367410
structure and assembly of an inner membrane platform for initiation of type iv pilus biogenesis.type iv pili are long fibers that are assembled by polymerization of a major pilin protein in the periplasm of a wide range of bacteria and archaea. they play crucial roles in pathogenesis, dna transformation, and motility, and are capable of rapid retraction, generating powerful motor forces. piln and pilo are integral inner membrane proteins that are essential for type iv pilus formation. here, we show that piln and pilo from thermus thermophilus can be isolated as a complex with pilm, a cytop ...201324218553
a structured-based model for the decreased activity of ala222val and glu429ala methylenetetrahydrofolate reductase (mthfr) mutants.the structure of human methylenetetrahydrofolate reductase (mthfr) is not known either by nmr or by x-ray methods. phosphorylation seems to play an important role in the functioning of this flavoprotein. mthfr catalyzes an irreversible reaction in homocysteine metabolism. phosphorylation decreases the activity of mthfr by enhancing the sensitivity of the enzyme to sadenosylmethione. two common polymorphisms in mthfr, ala222val and glu429ala, can result in a number of vascular diseases. effects o ...201324307772
architecture and gene repertoire of the flexible genome of the extreme acidophile acidithiobacillus caldus.acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the acidithiobacillales. as such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. in this study, we explore the genomic diversity of a. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome.201324250794
niche specialization of novel thaumarchaeota to oxic and hypoxic acidic geothermal springs of yellowstone national park.novel lineages of the phylum thaumarchaeota are endemic to thermal habitats, and may exhibit physiological capabilities that are not yet observed in members of this phylum. the primary goals of this study were to conduct detailed phylogenetic and functional analyses of metagenome sequence assemblies of two different thaumarchaeal populations found in high-temperature (65-72 °c), acidic (ph~3) iron oxide and sulfur sediment environments of yellowstone national park (ynp). metabolic reconstruction ...201324196321
niche specialization of novel thaumarchaeota to oxic and hypoxic acidic geothermal springs of yellowstone national park.novel lineages of the phylum thaumarchaeota are endemic to thermal habitats, and may exhibit physiological capabilities that are not yet observed in members of this phylum. the primary goals of this study were to conduct detailed phylogenetic and functional analyses of metagenome sequence assemblies of two different thaumarchaeal populations found in high-temperature (65-72 °c), acidic (ph~3) iron oxide and sulfur sediment environments of yellowstone national park (ynp). metabolic reconstruction ...201324196321
aminoacylation of plasmodium falciparum trna(asn) and insights in the synthesis of asparagine repeats.genome sequencing revealed an extreme at-rich genome and a profusion of asparagine repeats associated with low complexity regions (lcrs) in proteins of the malarial parasite plasmodium falciparum. despite their abundance, the function of these lcrs remains unclear. because they occur in almost all families of plasmodial proteins, the occurrence of lcrs cannot be associated with any specific metabolic pathway; yet their accumulation must have given selective advantages to the parasite. translatio ...201324196969
in silico insights of l-glutamate: structural features in vacuum and in complex with its receptor.structural properties of the glutamate in vacuum and in complex with its receptor were analyzed. the analysis was focused on global properties, attempting to characterize features such as overall flexibility and common trends in the conformation set. the glutamate, as other ligands in complex with the receptor, adopts a spatial conformation that corresponds to one of the possible molecular equilibrium states in physiological conditions. the glutamate forms an extended structure for all cases, bu ...201324307941
correlation between supercoiling and conformational motions of the bacterial flagellar filament.the bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. various supercoiled states of the filament exist, which are formed by two structurally different conformations of flagellin in different ratios. we investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. thermal fluctuations ...201324209861
rigidity analysis of protein biological assemblies and periodic crystal structures.we initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. the goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. we use experimental x-ray crystallography data from the protein data bank (pdb). the analysis relies on an effcient graph-based algorithm. computational experiments were performed using new protein rigidity analysis tools av ...201324564201
mechanisms of copper homeostasis in bacteria.copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. however, free copper is a potential hazard because of its high chemical reactivity. consequently, organisms exert a tight control on cu(+) transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a ...201324205499
sequence, structure, and stacking: specifics of trna anchoring to the t box riboswitch.the term riboswitch usually refers to small molecule sensing regulatory modules in the 5' untranslated regions of a mrna. they are typically comprised of separate ligand binding and regulatory domains. the t box riboswitch is unique from other identified riboswitches because its effector is an essential macromolecule, trna. it senses the aminoacylation state of trna to regulate genes involved in a variety of functions relating to amino acid metabolism and trna aminoacylation. t box riboswitches ...201324356646
insights into protein allostery in the csor/rcnr family of transcriptional repressors.csor/rcnr transcriptional repressors adopt a disc-shaped, all α-helical dimer of dimers tetrameric architecture, with a four-helix bundle the key structural feature of the dimer. individual members of this large family of repressors coordinate cu(i) or ni(ii)/co(ii) or perform cysteine sulfur chemistry in mitigating the effects of metal or metabolite toxicity, respectively. here we highlight recent insights into the functional diversity of this fascinating family of repressors.201324695963
insights into protein allostery in the csor/rcnr family of transcriptional repressors.csor/rcnr transcriptional repressors adopt a disc-shaped, all α-helical dimer of dimers tetrameric architecture, with a four-helix bundle the key structural feature of the dimer. individual members of this large family of repressors coordinate cu(i) or ni(ii)/co(ii) or perform cysteine sulfur chemistry in mitigating the effects of metal or metabolite toxicity, respectively. here we highlight recent insights into the functional diversity of this fascinating family of repressors.201324695963
Refresh
firstprev
...11121314151617181920...
nextlast
Displaying items 1201 - 1300 of 5451