Publications

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refining homology models by combining replica-exchange molecular dynamics and statistical potentials.a protocol is presented for the global refinement of homology models of proteins. it combines the advantages of temperature-based replica-exchange molecular dynamics (remd) for conformational sampling and the use of statistical potentials for model selection. the protocol was tested using 21 models. of these 14 were models of 10 small proteins for which high-resolution crystal structures were available, the remainder were targets of the recent caspr exercise. it was found that remd in combinatio ...200818338384
alignment of protein structures in the presence of domain motions.structural alignment is an important step in protein comparison. well-established methods exist for solving this problem under the assumption that the structures under comparison are considered as rigid bodies. however, proteins are flexible entities often undergoing movements that alter the positions of domains or subdomains with respect to each other. such movements can impede the identification of structural equivalences when rigid aligners are used.200818727838
redox regulation of protein folding in the mitochondrial intermembrane space.protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. however, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. the mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. specifically, the intermembrane space proteins with disulfide bonds are imported via th ...200818761382
redox regulation of protein folding in the mitochondrial intermembrane space.protein translocation pathways to the mitochondrial matrix and inner membrane have been well characterized. however, translocation into the intermembrane space, which was thought to be simply a modification of the traditional translocation pathways, is complex. the mechanism by which a subset of intermembrane space proteins, those with disulfide bonds, are translocated has been largely unknown until recently. specifically, the intermembrane space proteins with disulfide bonds are imported via th ...200818761382
conserved motifs in both cpsf73 and cpsf100 are required to assemble the active endonuclease for histone mrna 3'-end maturation.in eukaryotes, the process of messenger rna 3'-end formation involves endonucleolytic cleavage of the transcript followed by synthesis of the poly(a) tail. the complex machinery involved in this maturation process contains two proteins of the metallo-beta-lactamase (mbl) superfamily, the 73 and 100 kda subunits of the cleavage and polyadenylation specificity factor (cpsf). by using an in vitro system to assess point mutations in these two mammalian proteins, we found that conserved residues from ...200818688255
ppargamma and proline oxidase in cancer.proline is metabolized by its own specialized enzymes with their own tissue and subcellular localizations and mechanisms of regulation. the central enzyme in this metabolic system is proline oxidase, a flavin adenine dinucleotide-containing enzyme which is tightly bound to mitochondrial inner membranes. the electrons from proline can be used to generate atp or can directly reduce oxygen to form superoxide. although proline may be derived from the diet and biosynthesized endogenously, an importan ...200818670615
domain characterization of a 4-alpha-glucanotransferase essential for maltose metabolism in photosynthetic leaves.maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-alpha-glucanotransferase (ec 2.4.1.25) in the cytosol of leaf cells. this enzyme is called dpe2 because of its similarity to the disproportionating enzyme in plastids (dpe1). dpe1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. dpe2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar ...200818499663
structural frameworks for considering microbial protein- and nucleic acid-dependent motor atpases.many fundamental cellular processes depend on enzymes that utilize chemical energy to catalyse unfavourable reactions. certain classes of atpases provide a particularly vivid example of the process of energy conversion, employing cycles of nucleotide turnover to move and/or rearrange biological polymers such as proteins and nucleic acids. four well-characterized classes of atp-dependent protein/nucleic acid translocases and remodelling factors are found in all three domains of life (bacteria, ar ...200818647240
local function conservation in sequence and structure space.we assess the variability of protein function in protein sequence and structure space. various regions in this space exhibit considerable difference in the local conservation of molecular function. we analyze and capture local function conservation by means of logistic curves. based on this analysis, we propose a method for predicting molecular function of a query protein with known structure but unknown function. the prediction method is rigorously assessed and compared with a previously publis ...200818604264
amino acid modifications on trna.the accurate formation of cognate aminoacyl-transfer rnas (aa-trnas) is essential for the fidelity of translation. most amino acids are esterified onto their cognate trna isoacceptors directly by aa-trna synthetases. however, in the case of four amino acids (gln, asn, cys and sec), aminoacyl-trnas are made through indirect pathways in many organisms across all three domains of life. the process begins with the charging of noncognate amino acids to trnas by a specialized synthetase in the case of ...200818604446
mass spectrometry of the fifth nucleoside: a review of the identification of pseudouridine in nucleic acids.pseudouridine, the so-called fifth nucleoside due to its ubiquitous presence in ribonucleic acids (rnas), remains among the most challenging modified nucleosides to characterize. as an isomer of the major nucleoside uridine, pseudouridine cannot be detected by standard reverse-transcriptase-based dna sequencing or rnase mapping approaches. thus, over the past 15 years, investigators have focused on the unique structural properties of pseudouridine to develop selective derivatization or fragmenta ...200818620915
biophysical studies of bacterial ribosome assembly.the assembly of the bacterial ribosome involves the association of over 50 proteins to 3 large rna molecules, and it represents a major metabolic activity for rapidly growing bacteria. the availability of atomic structures of the ribosome and the application of biochemical and biophysical methods have led to rapid progress in understanding the mechanistic details of ribosome assembly. the basic steps required to assemble a ribosome are outlined, and the contributions of mass spectrometry, comput ...200818541423
aminoacyl-trna synthetase complexes: molecular multitasking revealed.the accurate synthesis of proteins, dictated by the corresponding nucleotide sequence encoded in mrna, is essential for cell growth and survival. central to this process are the aminoacyl-trna synthetases (aarss), which provide amino acid substrates for the growing polypeptide chain in the form of aminoacyl-trnas. the aarss are essential for coupling the correct amino acid and trna molecules, but are also known to associate in higher order complexes with proteins involved in processes beyond tra ...200818522650
compatible solute influence on nucleic acids: many questions but few answers.compatible solutes are small organic osmolytes including but not limited to sugars, polyols, amino acids, and their derivatives. they are compatible with cell metabolism even at molar concentrations. a variety of organisms synthesize or take up compatible solutes for adaptation to extreme environments. in addition to their protective action on whole cells, compatible solutes display significant effects on biomolecules in vitro. these include stabilization of native protein and nucleic acid struc ...200818522725
evolution of trna nucleotidyltransferases: a small deletion generated cc-adding enzymes.cca-adding enzymes are specialized polymerases that add a specific sequence (c-c-a) to trna 3' ends without requiring a nucleic acid template. in some organisms, cca synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (cc and a addition). these enzymes carry all known motifs of the catalytic core found in cca-adding enzymes. therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete ...200818523015
riboswitch effectors as protein enzyme cofactors.the recently identified glms ribozyme revealed that rna enzymes, like protein enzymes, are capable of using small molecules as catalytic cofactors to promote chemical reactions. flavin mononucleotide (fmn), s-adenosyl methionine (sam), adenosyl cobalamin (adocbl), and thiamine pyrophosphate (tpp) are known ligands for rna riboswitches in the control of gene expression, but are also catalytically powerful and ubiquitous cofactors in protein enzymes. if rna, instead of just binding these molecules ...200818430893
analysis of genomic trna sets from bacteria, archaea, and eukarya points to anticodon-codon hydrogen bonds as a major determinant of trna compositional variations.analysis of 100 complete sets of the cytoplasmic elongator trna genes from bacteria, archaea, and eukarya pointed to correspondences between types of anticodon and composition of the rest of the trna body. the number of the hydrogen bonds formed between the complementary nucleotides in the anticodon-codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. our analysis has supported and advanced the "extended anticodon" concept that is based o ...200818441051
viral ires rna structures and ribosome interactions.in eukaryotes, protein synthesis initiates primarily by a mechanism that requires a modified nucleotide 'cap' on the mrna and also proteins that recruit and position the ribosome. many pathogenic viruses use an alternative, cap-independent mechanism that substitutes rna structure for the cap and many proteins. the rnas driving this process are called internal ribosome-entry sites (iress) and some are able to bind the ribosome directly using a specific 3d rna structure. recent structures of ires ...200818468443
diversity surveys and evolutionary relationships of aoxb genes in aerobic arsenite-oxidizing bacteria.a new primer set was designed to specifically amplify ca. 1,100 bp of aoxb genes encoding the as(iii) oxidase catalytic subunit from taxonomically diverse aerobic as(iii)-oxidizing bacteria. comparative analysis of aoxb protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. aoxb phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16s rrna phylogeny. alphaproteobacteria-, betaproteo ...200818502920
cofactor dependent conformational switching of gtpases.this theoretical work covers structural and biochemical aspects of nucleotide binding and gdp/gtp exchange of gtp hydrolases belonging to the family of small gtpases. current models of gdp/gtp exchange regulation are often based on two specific assumptions. the first is that the conformation of a gtpase is switched by the exchange of the bound nucleotide from gdp to gtp or vice versa. the second is that gdp/gtp exchange is regulated by a guanine nucleotide exchange factor, which stabilizes a gtp ...200818502805
ribosomal position and contacts of mrna in eukaryotic translation initiation complexes.the position of mrna on 40s ribosomal subunits in eukaryotic initiation complexes was determined by uv crosslinking using mrnas containing uniquely positioned 4-thiouridines. crosslinking of mrna positions (+)11 to ribosomal protein (rp) rps2(s5p) and rps3(s3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18s rrna, respectively, indicated that mrna enters the mrna-binding channel through the same layers of rrna and proteins as in prokaryotes. upstream of the p-site, the proximity of positions ...200818464793
architectures and functional coverage of protein-protein interfaces.the diverse range of cellular functions is performed by a limited number of protein folds existing in nature. one may similarly expect that cellular functional diversity would be covered by a limited number of protein-protein interface architectures. here, we present 8205 interface clusters, each representing a unique interface architecture. this data set of protein-protein interfaces is analyzed and compared with older data sets. we observe that the number of both biological and crystal interfa ...200818620705
molecular design of beta-hairpin peptides for material construction.self-assembled materials composed of beta-sheet forming peptides hold promise as therapeutics and novel biomaterials. this article focuses on the design and engineering of amphiphilic peptide sequences, especially beta-hairpins. peptides can be designed to intramolecularly fold and then self-assemble on cue, affording hydrogels rich in beta-sheet structure. hydrogels having distinct material properties can be designed at the molecular level by modulating either the peptide's sequence or the envi ...200819662109
the aaa+ superfamily of functionally diverse proteins.the aaa+ superfamily is a large and functionally diverse superfamily of ntpases that are characterized by a conserved nucleotide-binding and catalytic module, the aaa+ module. members are involved in an astonishing range of different cellular processes, attaining this functional diversity through additions of structural motifs and modifications to the core aaa+ module.200818466635
the nod-like receptor (nlr) family: a tale of similarities and differences.innate immunity represents an important system with a variety of vital processes at the core of many diseases. in recent years, the central role of the nod-like receptor (nlr) protein family became increasingly appreciated in innate immune responses. nlrs are classified as part of the signal transduction atpases with numerous domains (stand) clade within the aaa+ atpase family. they typically feature an n-terminal effector domain, a central nucleotide-binding domain (nacht) and a c-terminal liga ...200818446235
the past and present of sodium energetics: may the sodium-motive force be with you.all living cells routinely expel na(+) ions, maintaining lower concentration of na(+) in the cytoplasm than in the surrounding milieu. in the vast majority of bacteria, as well as in mitochondria and chloroplasts, export of na(+) occurs at the expense of the proton-motive force. some bacteria, however, possess primary generators of the transmembrane electrochemical gradient of na(+) (sodium-motive force). these primary na(+) pumps have been traditionally seen as adaptations to high external ph o ...200818485887
dual-targeted trna-dependent amidotransferase ensures both mitochondrial and chloroplastic gln-trnagln synthesis in plants.aminoacyl-trnas are generally formed by direct attachment of an amino acid to trnas by aminoacyl-trna synthetases, but gln-trna is an exception to this rule. gln-trna(gln) is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. we show here that the formation of gln-trna(gln) is also achieved by the indirect pathway in plant mitochondria. the mitochondrial- ...200818441100
the origin and evolution of the ribosome.the origin and early evolution of the active site of the ribosome can be elucidated through an analysis of the ribosomal proteins' taxonomic block structures and their rna interactions. comparison between the two subunits, exploiting the detailed three-dimensional structures of the bacterial and archaeal ribosomes, is especially informative.200818430223
association of aminoglycosidic antibiotics with the ribosomal a-site studied with brownian dynamics.brownian dynamics methodology was applied to simulate the encounter of aminoglycosidic antibiotics with the ribosomal a-site rna. studied antibiotics included neamine, neomycin, ribostamycin and paromomycin which differ in chemical structure, the number of pseudo-sugar rings and the net charge. the influence of structural, electrostatic and hydrodynamic properties of antibiotics on the kinetics of their association with the ribosomal a-site was analyzed. the computed diffusion limited rates of a ...200819343095
whep domains direct noncanonical function of glutamyl-prolyl trna synthetase in translational control of gene expression.the heterotetrameric gait complex suppresses translation of selected mrnas in interferon-gamma-activated monocytic cells. specificity is dictated by glutamyl-prolyl trna synthetase (eprs) binding to a 3'utr element in target mrnas. eprs consists of two synthetase cores joined by a linker containing three whep domains of unknown function. here we show the critical role of eprs whep domains in targeting and regulating gait complex binding to rna. the upstream whep pair directs high-affinity bindin ...200818374644
estimating the fraction of non-coding rnas in mammalian transcriptomes.recent studies of mammalian transcriptomes have identified numerous rna transcripts that do not code for proteins; their identity, however, is largely unknown. here we explore an approach based on sequence randomness patterns to discern different rna classes. the relative z-score we use helps identify the known ncrna class from the genome, intergene and intron classes. this leads us to a fractional ncrna measure of putative ncrna datasets which we model as a mixture of genuine ncrnas and other t ...200819812767
mitochondrial deafness alleles confer misreading of the genetic code.despite the fact that important genetic diseases are caused by mutant mitochondrial ribosomes, the molecular mechanisms by which such ribosomes result in a clinical phenotype remain largely unknown. the absence of experimental models for mitochondrial diseases has also prevented the rational search for therapeutic interventions. here, we report on the construction of bacterial hybrid ribosomes that contain various versions of the mitochondrial decoding region of ribosomal rna. we show that the p ...200818308926
translational attenuation and mrna stabilization as mechanisms of erm(b) induction by erythromycin.translational attenuation has been proposed to be the mechanism by which the erm(b) gene is induced. here, we report genetic and biochemical evidence, obtained by using erythromycin as the inducing antibiotic, that supports this hypothesis. we also show that erythromycin increases the level of the erm(b) transcript by stalling the ribosome on the leader mrna and thereby facilitating the stabilization and processing of the mrna. erythromycin-induced mrna stabilization and processing were observed ...200818299414
from one amino acid to another: trna-dependent amino acid biosynthesis.aminoacyl-trnas (aa-trnas) are the essential substrates for translation. most aa-trnas are formed by direct aminoacylation of trna catalyzed by aminoacyl-trna synthetases. however, a smaller number of aa-trnas (asn-trna, gln-trna, cys-trna and sec-trna) are made by synthesizing the amino acid on the trna by first attaching a non-cognate amino acid to the trna, which is then converted to the cognate one catalyzed by trna-dependent modifying enzymes. asn-trna or gln-trna formation in most prokaryo ...200818252769
protein structure fitting and refinement guided by cryo-em density.for many macromolecular assemblies, both a cryo-electron microscopy map and atomic structures of its component proteins are available. here we describe a method for fitting and refining a component structure within its map at intermediate resolution (<15 a). the atomic positions are optimized with respect to a scoring function that includes the crosscorrelation coefficient between the structure and the map as well as stereochemical and nonbonded interaction terms. a heuristic optimization that r ...200818275820
assays for transfer rna-dependent amino acid biosynthesis.selenocysteinyl-trna(sec), cysteinyl-trna(cys), glutaminyl-trna(gln), and asparaginyl-trna(asn) in many organisms are formed in an indirect pathway in which a non-cognate amino acid is first attached to the trna. this non-cognate amino acid is then converted to the cognate amino acid by a trna-dependent modifying enzyme. the in vitro characterization of these modifying enzymes is challenging due to the fact the substrate, aminoacyl-trna, is labile and requires a prior enzymatic step to be synthe ...200818241795
functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.cysteine sulfenic acid (cys-soh), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. this post-translational modification is not random: specific features near the cysteine control its reactivity. to identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known cys-soh sites) was compi ...200818227433
molecular adaptation and expression evolution following duplication of genes for organellar ribosomal protein s13 in rosids.gene duplication has been a fundamental process in the evolution of eukaryotic genomes. after duplication one copy (or both) can undergo divergence in sequence, expression pattern, and function. two divergent copies of the ribosomal protein s13 gene (rps13) of chloroplast origin are found in the nucleus of the rosids arabidopsis, gossypium, and glycine. one encodes chloroplast-imported rps13 (nucp rps13), while the other encodes mitochondria-imported rps13 (numit rps13). the rps13 gene has been ...200818221556
chaperones in control of protein disaggregation.the chaperone protein network controls both initial protein folding and subsequent maintenance of proteins in the cell. although the native structure of a protein is principally encoded in its amino-acid sequence, the process of folding in vivo very often requires the assistance of molecular chaperones. chaperones also play a role in a post-translational quality control system and thus are required to maintain the proper conformation of proteins under changing environmental conditions. many fact ...200818216875
on the evolution of the trna-dependent amidotransferases, gatcab and gatde.glutaminyl-trna synthetase and asparaginyl-trna synthetase evolved from glutamyl-trna synthetase and aspartyl-trna synthetase, respectively, after the split in the last universal communal ancestor (luca). glutaminyl-trna(gln) and asparaginyl-trna(asn) were likely formed in luca by amidation of the mischarged species, glutamyl-trna(gln) and aspartyl-trna(asn), by trna-dependent amidotransferases, as is still the case in most bacteria and all known archaea. the amidotransferase gatcab is found in ...200818279892
the elongation factor rfah and the initiation factor sigma bind to the same site on the transcription elongation complex.rna polymerase is a target for numerous regulatory events in all living cells. recent studies identified a few "hot spots" on the surface of bacterial rna polymerase that mediate its interactions with diverse accessory proteins. prominent among these hot spots, the beta' subunit clamp helices serve as a major binding site for the initiation factor sigma and for the elongation factor rfah. furthermore, the two proteins interact with the nontemplate dna strand in transcription complexes and thus m ...200818195372
structural biology of riboswitch-mediated gene regulation and argonaute-mediated gene silencing. 200818776223
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
bacterial heme-transport proteins and their heme-coordination modes.efficient iron acquisition is critical for an invading microbe's survival and virulence. most of the iron in mammals is incorporated into heme, which can be plundered by certain bacterial pathogens as a nutritional iron source. utilization of exogenous heme by bacteria involves the binding of heme or hemoproteins to the cell surface receptors, followed by the transport of heme into cells. once taken into the cytosol, heme is presented to heme oxygenases where the tetrapyrrole ring is cleaved in ...200818977196
the elusive object of desire--interactions of bacteriophages and their hosts.bacteria and their viruses (phages) are locked in an evolutionary contest, with each side producing constantly changing mechanisms of attack and defense that are aimed to increase the odds of survival. as a result, phages play central roles in a great variety of genetic processes and increase the rate of evolutionary change of the bacterial host, which could ultimately work to the benefit of the host in a long run.200818400552
structural biology of proline catabolism.the proline catabolic enzymes proline dehydrogenase and delta(1)-pyrroline-5-carboxylate dehydrogenase catalyze the 4-electron oxidation of proline to glutamate. these enzymes play important roles in cellular redox control, superoxide generation, apoptosis and cancer. in some bacteria, the two enzymes are fused into the bifunctional enzyme, proline utilization a. here we review the three-dimensional structural information that is currently available for proline catabolic enzymes. crystal structu ...200818369526
tie me up, tie me down: inhibiting rna polymerase.mechanistic understanding of antibiotic action can yield crucial insights that aid in the design of new antibiotics. in this issue, mukhopadhyay et al. (2008) uncover the mechanism by which the antibiotic myxopyronin inhibits bacterial rna polymerase, suggesting a new target region in rna polymerase for inhibitor design.200818957193
soda: an mn/fe superoxide dismutase prediction and design server.superoxide dismutases (sods) are ubiquitous metalloenzymes that play an important role in the defense of aerobic organisms against oxidative stress, by converting reactive oxygen species into nontoxic molecules. we focus here on the sod family that uses fe or mn as cofactor.200818518943
recognition of aminoacyl-trna: a common molecular mechanism revealed by cryo-em.the accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by ef-tu, which forms a ternary complex with aminoacyl(aa)-trna. to study the binding modes of different aa-trnas, we compared cryo-em maps of the kirromycin-stalled ribosome bound with ternary complexes containing phe-trna(phe), trp-trna(trp), or leu-trna(leui). the three maps suggest a common binding manner of cognate aa-trnas in their specific binding with both the ribosome and ef-tu. a ...200819020518
organization of an activator-bound rna polymerase holoenzyme.transcription initiation involves the conversion from closed promoter complexes, comprising rna polymerase (rnap) and double-stranded promoter dna, to open complexes, in which the enzyme is able to access the dna template in a single-stranded form. the complex between bacterial rnap and its major variant sigma factor sigma(54) remains as a closed complex until atp hydrolysis-dependent remodeling by activator proteins occurs. this remodeling facilitates dna melting and allows the transition to th ...200818995832
rifamycins do not function by allosteric modulation of binding of mg2+ to the rna polymerase active center.rifamycin antibacterial agents inhibit bacterial rna polymerase (rnap) by binding to a site adjacent to the rnap active center and preventing synthesis of rna products >2-3 nt in length. recently, artsimovitch et al. [(2005) cell 122:351-363] proposed that rifamycins function by allosteric modulation of binding of mg(2+) to the rnap active center and presented three lines of biochemical evidence consistent with this proposal. here, we show that rifamycins do not affect the affinity of binding of ...200818787125
enzymes used in molecular biology: a useful guide.since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. this review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. we provide ...200818766469
advances in bacterial promoter recognition and its control by factors that do not bind dna.early work identified two promoter regions, the -10 and -35 elements, that interact sequence specifically with bacterial rna polymerase (rnap). however, we now know that several additional promoter elements contact rnap and influence transcription initiation. furthermore, our picture of promoter control has evolved beyond one in which regulation results solely from activators and repressors that bind to dna sequences near the rnap binding site: many important transcription factors bind directly ...200818521075
on helicases and other motor proteins.helicases are molecular machines that utilize energy derived from atp hydrolysis to move along nucleic acids and to separate base-paired nucleotides. the movement of the helicase can also be described as a stationary helicase that pumps nucleic acid. recent structural data for the hexameric e1 helicase of papillomavirus in complex with single-stranded dna and mgadp has provided a detailed atomic and mechanistic picture of its atp-driven dna translocation. the structural and mechanistic features ...200818329872
"hot cores" in proteins: comparative analysis of the apolar contact area in structures from hyper/thermophilic and mesophilic organisms.a wide variety of stabilizing factors have been invoked so far to elucidate the structural basis of protein thermostability. these include, amongst the others, a higher number of ion-pairs interactions and hydrogen bonds, together with a better packing of hydrophobic residues. it has been frequently observed that packing of hydrophobic side chains is improved in hyperthermophilic proteins, when compared to their mesophilic counterparts. in this work, protein crystal structures from hyper/thermop ...200818312638
cytochrome c oxidase: exciting progress and remaining mysteries.cytochrome c oxidase generates a proton motive force by two separate mechanisms. the first mechanism is similar to that postulated by peter mitchell, and is based on electrons and protons used to generate water coming from opposite sides of the membrane. the second mechanism was not initially anticipated, but is now firmly established as a proton pump. a brief review of the current state of our understanding of the proton pump of cytochrome oxidase is presented. we have come a long way since the ...200818975062
the chemistry and biochemistry of heme c: functional bases for covalent attachment.a discussion of the literature concerning the synthesis, function, and activity of heme c-containing proteins is presented. comparison of the properties of heme c, which is covalently bound to protein, is made to heme b, which is bound noncovalently. a question of interest is why nature uses biochemically expensive heme c in many proteins when its properties are expected to be similar to heme b. considering the effects of covalent heme attachment on heme conformation and on the proximal histidin ...200819030605
one heme, diverse functions: using biosynthetic myoglobin models to gain insights into heme-copper oxidases and nitric oxide reductases. 200818729107
the q-cycle reviewed: how well does a monomeric mechanism of the bc(1) complex account for the function of a dimeric complex?recent progress in understanding the q-cycle mechanism of the bc(1) complex is reviewed. the data strongly support a mechanism in which the q(o)-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron-sulfur protein is the rate-determining step for the overall process. the reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2fe-2s] cluster, in which the unfavorable protoni ...200818501698
nadh/nad+ interaction with nadh: ubiquinone oxidoreductase (complex i).the quantitative data on the binding affinity of nadh, nad(+), and their analogues for complex i as emerged from the steady-state kinetics data and from more direct studies under equilibrium conditions are summarized and discussed. the redox-dependency of the nucleotide binding and the reductant-induced change of fmn affinity to its tight non-covalent binding site indicate that binding (dissociation) of the substrate (product) may energetically contribute to the proton-translocating activity of ...200818471432
function-biased choice of additives for optimization of protein crystallization - the case of the putative thioesterase pa5185 from pseudomonas aeruginosa pao1.the crystal structure of pa5185, a putative thioesterase from pseudomonas aeruginosa strain pao1, was solved using multi-wavelength anomalous diffraction to 2.4 a. analysis of the structure and information about the putative function of the protein were used to optimize crystallization conditions. the crystal growth was optimized by applying additives with chemical similarity to a fragment of a putative pa5185 substrate (coa or its derivative). using new crystallization conditions containing thi ...200819898606
structure of an argonaute silencing complex with a seed-containing guide dna and target rna duplex.here we report on a 3.0 a crystal structure of a ternary complex of wild-type thermus thermophilus argonaute bound to a 5'-phosphorylated 21-nucleotide guide dna and a 20-nucleotide target rna containing cleavage-preventing mismatches at the 10-11 step. the seed segment (positions 2 to 8) adopts an a-helical-like watson-crick paired duplex, with both ends of the guide strand anchored in the complex. an arginine, inserted between guide-strand bases 10 and 11 in the binary complex, locking it in a ...200819092929
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
large deviations for random trees and the branching of rna secondary structures.we give a large deviation principle (ldp) with explicit rate function for the distribution of vertex degrees in plane trees, a combinatorial model of rna secondary structures. we calculate the typical degree distributions based on nearest neighbor free energies, and compare our results with the branching configurations found in two sets of large rna secondary structures. we find substantial agreement overall, with some interesting deviations which merit further study.200819083065
genomics of bacteria and archaea: the emerging dynamic view of the prokaryotic world.the first bacterial genome was sequenced in 1995, and the first archaeal genome in 1996. soon after these breakthroughs, an exponential rate of genome sequencing was established, with a doubling time of approximately 20 months for bacteria and approximately 34 months for archaea. comparative analysis of the hundreds of sequenced bacterial and dozens of archaeal genomes leads to several generalizations on the principles of genome organization and evolution. a crucial finding that enables function ...200818948295
minimum contradiction matrices in whole genome phylogenies.minimum contradiction matrices are a useful complement to distance-based phylogenies. a minimum contradiction matrix represents phylogenetic information under the form of an ordered distance matrix y(i) (,) (j) (n). a matrix element corresponds to the distance from a reference vertex n to the path (i, j). for an x-tree or a split network, the minimum contradiction matrix is a robinson matrix. it therefore fulfills all the inequalities defining perfect order: y(i) (,) (j) (n) >or= y(i) (,) (k) (n ...200819204821
evolutionary primacy of sodium bioenergetics.the f- and v-type atpases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of atp. both the f-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and v-type (found in archaea, some bacteria, and eukaryotic vacuoles) atpases can translocate either protons or sodium ions. the prevalent proton-dependent atpases are generally viewed as the primary form of the enzyme whereas the sodium-transloca ...200818380897
the application of fast-nmr for the identification of novel drug discovery targets.the continued success of genome sequencing projects has resulted in a wealth of information, but 40-50% of identified genes correspond to hypothetical proteins or proteins of unknown function. the functional annotation screening technology by nmr (fast-nmr) screen was developed to assign a biological function for these unannotated proteins with a structure solved by the protein structure initiative. fast-nmr is based on the premise that a biological function can be described by a similarity in b ...200818275915
revealing unique properties of the ribosome using a network based analysis.the ribosome is a complex molecular machine that offers many potential sites for functional interference, therefore representing a major target for antibacterial drugs. the growing number of high-resolution structures of ribosomes from different organisms, in free form and in complex with various ligands, provides unique data for structural and comparative analyses of rna structures. we model the ribosome structure as a network, where nucleotides are represented as nodes and intermolecular inter ...200818625614
conserved discrimination against misacylated trnas by two mesophilic elongation factor tu orthologs.elongation factor tu (ef-tu) binds and loads elongating aminoacyl-trnas (aa-trnas) onto the ribosome for protein biosynthesis. many bacteria biosynthesize gln-trna (gln) and asn-trna (asn) by an indirect, two-step pathway that relies on the misacylated trnas glu-trna (gln) and asp-trna (asn) as intermediates. previous thermodynamic and experimental analyses have demonstrated that thermus thermophilus ef-tu does not bind asp-trna (asn) and predicted a similar discriminatory response against glu-t ...200818627126
the complete genome sequence of moorella thermoacetica (f. clostridium thermoaceticum).this paper describes the genome sequence of moorella thermoacetica (f. clostridium thermoaceticum), which is the model acetogenic bacterium that has been widely used for elucidating the wood-ljungdahl pathway of co and co(2) fixation. this pathway, which is also known as the reductive acetyl-coa pathway, allows acetogenic (often called homoacetogenic) bacteria to convert glucose stoichiometrically into 3 mol of acetate and to grow autotrophically using h(2) and co as electron donors and co(2) as ...200818631365
molecular mechanisms underlying the positive stringent response of the bacillus subtilis ilv-leu operon, involved in the biosynthesis of branched-chain amino acids.branched-chain amino acids are the most abundant amino acids in proteins. the bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. this operon exhibits a rela-dependent positive stringent response to amino acid starvation. we investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. deletion analysis involving various lacz fusions revealed two molecular mechanisms underlying the positive stringent response of ...200818641142
erythromycin-induced ribosome stalling and rnase j1-mediated mrna processing in bacillus subtilis.summary: addition of erythromycin (em) to a bacillus subtilis strain carrying the ermc gene results in ribosome stalling in the ermc leader peptide coding sequence. using deltaermc, a deletion derivative of ermc that specifies the 254 nucleotide deltaermc mrna, we showed previously that ribosome stalling is concomitant with processing of deltaermc mrna, generating a 209 nucleotide rna whose 5' end maps to codon 5 of the deltaermc coding sequence. here we probed for peptidyl-trna to show that rib ...200818647167
the highest affinity binding site of small protein b on transfer messenger rna is outside the trna domain.eubacterial ribosomes stalled on defective mrnas are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein b (smpb) and transfer messenger rna (tmrna). a series of tmrna variants with deletions in each structural domain were produced. their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of smpb. dissociation constants between these rnas and smpb from aquifex aeolicus were derived by s ...200818648069
a new regulatory circuit in ribosomal protein operons: s2-mediated control of the rpsb-tsf expression in vivo.autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsb-tsf encoding essential r-protein s2 and elongation factor ts, where even the promoter has remained unknown. by using single-copy translational fusions with the chromosomal lacz gene and western-blot analysis, we demonstrate here that s2 serves as a negative regulator of both rpsb and tsf expression ...200818648071
crenarchaeal arginine decarboxylase evolved from an s-adenosylmethionine decarboxylase enzyme.the crenarchaeon sulfolobus solfataricus uses arginine to produce putrescine for polyamine biosynthesis. however, genome sequences from s. solfataricus and most crenarchaea have no known homologs of the previously characterized pyridoxal 5'-phosphate or pyruvoyl-dependent arginine decarboxylases that catalyze the first step in this pathway. instead they have two paralogs of the s-adenosylmethionine decarboxylase (adometdc). the gene at locus sso0585 produces an adometdc enzyme, whereas the gene ...200818650422
structure and mechanistic implications of a uroporphyrinogen iii synthase-product complex.uroporphyrinogen iii synthase (u3s) catalyzes the asymmetrical cyclization of a linear tetrapyrrole to form the physiologically relevant uroporphyrinogen iii (uro'gen iii) isomer during heme biosynthesis. here, we report four apoenzyme and one product complex crystal structures of the thermus thermophilus (hb27) u3s protein. the overlay of eight crystallographically unique u3s molecules reveals a huge range of conformational flexibility, including a "closed" product complex. the product, uro'gen ...200818651750
distinct double- and single-stranded dna binding of e. coli replicative dna polymerase iii alpha subunit.the alpha subunit of the replicative dna polymerase iii of escherichia coli is the active polymerase of the 10-subunit bacterial replicase. the c-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (ob-fold) domain. in a series of optical tweezers experiments, the alpha subunit is shown to have an affinity for both double- and single-stranded dna, in distinct subdomains of the protein. the portion of the protein that binds to double-stranded dna stabilizes the ...200818652472
origin of the nucleus and ran-dependent transport to safeguard ribosome biogenesis in a chimeric cell.the origin of the nucleus is a central problem about the origin of eukaryotes. the common ancestry of nuclear pore complexes (npc) and vesicle coating complexes indicates that the nucleus evolved via the modification of a pre-existing endomembrane system. such an autogenous scenario is cell biologically feasible, but it is not clear what were the selective or neutral mechanisms that had led to the origin of the nuclear compartment.200818652645
a unique conformation of the anticodon stem-loop is associated with the capacity of trnafmet to initiate protein synthesis.in all organisms, translational initiation takes place on the small ribosomal subunit and two classes of methionine trna are present. the initiator is used exclusively for initiation of protein synthesis while the elongator is used for inserting methionine internally in the nascent polypeptide chain. the crystal structure of escherichia coli initiator trna(f)(met) has been solved at 3.1 a resolution. the anticodon region is well-defined and reveals a unique structure, which has not been describe ...200818653533
recr forms a ring-like tetramer that encircles dsdna by forming a complex with recf.in the recfor pathway, the recf and recr proteins form a complex that binds to dna and exerts multiple functions, including directing the loading of reca onto single-stranded (ss) dna regions near double-stranded (ds) dna-ssdna junctions and preventing it from forming a filament beyond the ssdna region. however, neither the structure of the recfr complex nor its dna-binding mechanism was previously identified. here, size-exclusion chromatography and small-angle x-ray scattering data indicate tha ...200818658243
crystal structure of the thermus thermophilus 16 s rrna methyltransferase rsmc in complex with cofactor and substrate guanosine.post-transcriptional modification is a ubiquitous feature of ribosomal rna in all kingdoms of life. modified nucleotides are generally clustered in functionally important regions of the ribosome, but the functional contribution to protein synthesis is not well understood. here we describe high resolution crystal structures for the n(2)-guanine methyltransferase rsmc that modifies residue g1207 in 16 s rrna near the decoding site of the 30 s ribosomal subunit. rsmc is a class i s-adenosyl-l-methi ...200818667428
the rna acetyltransferase driven by atp hydrolysis synthesizes n4-acetylcytidine of trna anticodon.the wobble base of escherichia coli elongator trna(met) is modified to n(4)-acetylcytidine (ac(4)c), which is thought to ensure the precise recognition of the aug codon by preventing misreading of near-cognate aua codon. by employing genome-wide screen of uncharacterized genes in escherichia coli ('ribonucleome analysis'), we found the ypfi gene, which we named tmca (trna(met) cytidine acetyltransferase), to be responsible for ac(4)c formation. tmca is an enzyme that contains a walker-type atpas ...200818668122
thermodynamic redox behavior of the heme centers in a-type heme-copper oxygen reductases: comparison between the two subfamilies.the study of the thermodynamic redox behavior of the hemes from two members of the a family of heme-copper oxygen reductases, paracoccus denitrificans aa3 (a1 subfamily) and rhodothermus marinus caa3 (a2 subfamily) enzymes, is presented. at different ph values, midpoint reduction potentials and interaction potentials were obtained in the framework of a pairwise model for two interacting redox centers. in both enzymes, the hemes have different reduction potentials. for the a1-type enzyme, it was ...200818676644
deciphering the genetic determinants for aerobic nicotinic acid degradation: the nic cluster from pseudomonas putida kt2440.the aerobic catabolism of nicotinic acid (na) is considered a model system for degradation of n-heterocyclic aromatic compounds, some of which are major environmental pollutants; however, the complete set of genes as well as the structural-functional relationships of most of the enzymes involved in this process are still unknown. we have characterized a gene cluster (nic genes) from pseudomonas putida kt2440 responsible for the aerobic na degradation in this bacterium and when expressed in heter ...200818678916
cloning, expression, purification and preliminary crystallographic analysis of the rnase hi domain of the mycobacterium tuberculosis protein rv2228c as a maltose-binding protein fusion.the predicted ribonuclease (rnase) hi domain of the open reading frame rv2228c from mycobacterium tuberculosis has been cloned as a hexahistidine fusion and a maltose-binding protein (mbp) fusion. expression was only observed for the mbp-fusion protein, which was purified using amylose affinity chromatography and gel filtration. the rnase hi domain could be cleaved from the mbp-fusion protein by factor xa digestion, but was very unstable. in contrast, the fusion protein was stable, could be obta ...200818678948
structure of the e. coli dna glycosylase alka bound to the ends of duplex dna: a system for the structure determination of lesion-containing dna.the constant attack on dna by endogenous and exogenous agents gives rise to nucleobase modifications that cause mutations, which can lead to cancer. visualizing the effects of these lesions on the structure of duplex dna is key to understanding their biologic consequences. the most definitive method of obtaining such structures, x-ray crystallography, is troublesome to employ owing to the difficulty of obtaining diffraction-quality crystals of dna. here, we present a crystallization system that ...200818682218
hyperthermophilic aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines.the placement of the extreme thermophile aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. we investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically dnag primase. although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for a. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. to determi ...200818684998
the growth-promoting and stress response activities of the bacillus subtilis gtp binding protein obg are separable by mutation.bacillus subtilis obg is a ribosome-associating gtp binding protein that is needed for growth, sporulation, and induction of the bacterium's general stress regulon (gsr). it is unclear whether the roles of obg in sporulation and stress responsiveness are direct or a secondary effect of its growth-promoting functions. the present work addresses this question by an analysis of two obg alleles whose phenotypes argue for direct roles for obg in each process. the first allele [obg(g92d)] encodes a mi ...200818689482
insights into the replisome from the structure of a ternary complex of the dna polymerase iii alpha-subunit.the crystal structure of the catalytic alpha-subunit of the dna polymerase iii (pol iiialpha) holoenzyme bound to primer-template dna and an incoming deoxy-nucleoside 5'-triphosphate has been determined at 4.6-a resolution. the polymerase interacts with the sugar-phosphate backbone of the dna across its minor groove, which is made possible by significant movements of the thumb, finger, and beta-binding domains relative to their orientations in the unliganded polymerase structure. additionally, t ...200818691598
crystal structure of type 2 isopentenyl diphosphate isomerase from thermus thermophilus in complex with inorganic pyrophosphate.the n-terminal region is stabilized in the crystal structure of thermus thermophilus type 2 isopentenyl diphosphate isomerase in complex with inorganic pyrophosphate, providing new insights about the active site and the catalytic mechanism of the enzyme. the pp i moiety is located near the conserved residues, h10, r97, h152, q157, e158, and w219, and the flavin cofactor. the putative active site of isopentenyl diphosphate isomerase 2 provides interactions for stabilizing a carbocationic intermed ...200818693754
crystal structures of nadh:fmn oxidoreductase (emob) at different stages of catalysis.edta has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. recently, two critical enzymes, edta monooxygenase (emoa) and nadh:fmn oxidoreductase (emob), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the edta degradation pathway in mesorhizobium sp. bnc1. emoa is an fmnh2-dependent enzyme that requires emob to provide fmnh2 for the conversion of edta to ethylenediaminedia ...200818701448
mitochondrial nadh fluorescence is enhanced by complex i binding.mitochondrial nadh fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. mitochondrial nadh fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (efl). however, the actual binding sites responsible for nadh efl are unknown. we tested the hypothesis that nadh binding to complex i is a significant source of mitochondrial nadh fluorescence enhancement. to test this hypothesis, the effect of complex i binding on n ...200818702505
genetic identification of yeast 18s rrna residues required for efficient recruitment of initiator trna(met) and aug selection.high-resolution structures of bacterial 70s ribosomes have provided atomic details about mrna and trna binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (pics). we identified residues in yeast 18s rrna critical in vivo for recruiting methionyl trna(i)(met) to 40s subunits during initiation by isolating mutations that derepress gcn4 mrna translation. several such gcd(-) mutations alter the a928:u1389 base pair in helix 28 (h28) and allow ...200818708582
slr1923 of synechocystis sp. pcc6803 is essential for conversion of 3,8-divinyl(proto)chlorophyll(ide) to 3-monovinyl(proto)chlorophyll(ide).the deduced amino acid sequence of an slr1923 gene of synechocystis sp. pcc6803 is homologous to archaean f(420)h(2) dehydrogenase, which acts as a soluble subcomplex of reduced nicotinamide adenine dinucleotide dehydrogenase complex i. in this study, the gene was inactivated and characteristics of the mutant were analyzed. the mutant grew slower than the wild type under 100 microe m(-2) s(-1) but did not grow under high light intensity (300 microe m(-2) s(-1)). the cellular content of chlorophy ...200818715956
s-adenosyl-l-methionine hydrolase (adenosine-forming), a conserved bacterial and archaeal protein related to sam-dependent halogenases. 200818720493
purification and characterization of the bacterial udp-glcnac:undecaprenyl-phosphate glcnac-1-phosphate transferase weca.to date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate n-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. in the present work and for the first time, the integral membrane protein weca that catalyzes the transfer of the glcnac-1-phosphate moiety from udp-glcnac onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl ...200818723618
the rate and character of spontaneous mutation in thermus thermophilus.selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrf and pyre) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium thermus thermophilus. two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. the genomic mutation rate estimated from these targets, 0.00097 +/- 0.0 ...200818723895
the d subunit plays a central role in human vacuolar h(+)-atpases.the multi-subunit vacuolar-type h(+)-atpase consists of a v(1) domain (a-h subunits) catalyzing atp hydrolysis and a v(0) domain (a, c, c', c", d, e) responsible for h(+) translocation. the mammalian v(0) d subunit is one of the least-well characterized, and its function and position within the pump are still unclear. it has two different forms encoded by separate genes, d1 being ubiquitous while d2 is predominantly expressed at the cell surface in kidney and osteoclast. to determine whether it ...200818752060
electron and proton transfer in the ba(3) oxidase from thermus thermophilus.the ba(3)-type cytochrome c oxidase from thermus thermophilus is phylogenetically very distant from the aa(3)-type cytochrome c oxidases. nevertheless, both types of oxidases have the same number of redox-active metal sites and the reduction of o(2) to water is catalysed at a haem a(3)-cu(b) catalytic site. the three-dimensional structure of the ba(3) oxidase reveals three possible proton-conducting pathways showing very low homology compared to those of the mitochondrial, rhodobacter sphaeroide ...200818752061
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