Publications

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efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature.an efficient optimization method for the crystallization of biological macromolecules has been developed and tested. this builds on a successful high-throughput technique for the determination of initial crystallization conditions. the optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. the amount of sample and number of steps is minimized and no biochem ...200717327388
diversity and abundance of nitrate reductase genes (narg and napa), nitrite reductase genes (nirs and nrfa), and their transcripts in estuarine sediments.estuarine systems are the major conduits for the transfer of nitrate from agricultural and other terrestrial-anthropogenic sources into marine ecosystems. within estuarine sediments some microbially driven processes (denitrification and anammox) result in the net removal of nitrogen from the environment, while others (dissimilatory nitrate reduction to ammonium) do not. in this study, molecular approaches have been used to investigate the diversity, abundance, and activity of the nitrate-reducin ...200717400770
nanodiscs unravel the interaction between the secyeg channel and its cytosolic partner seca.the translocon is a membrane-embedded protein assembly that catalyzes protein movement across membranes. the core translocon, the secyeg complex, forms oligomers, but the protein-conducting channel is at the center of the monomer. defining the properties of the secyeg protomer is thus crucial to understand the underlying function of oligomerization. we report here the reconstitution of a single secyeg complex into nano-scale lipid bilayers, termed nanodiscs. these water-soluble particles allow o ...200717396152
environment specific substitution tables for thermophilic proteins.thermophilic organisms are able to live at high temperatures ranging from 50 to > 100 degrees c. their proteins must be sufficiently stable to function under these extreme conditions; however, the basis for thermostability remains elusive. subtle differences between thermophilic and mesophilic molecules can be found when sequences or structures from homologous proteins are compared, but often these differences are family-specific and few general rules have been derived. the availability of compl ...200717430559
structural and evolutionary bioinformatics of the spout superfamily of methyltransferases.spout methyltransferases (mtases) are a large class of s-adenosyl-l-methionine-dependent enzymes that exhibit an unusual alpha/beta fold with a very deep topological knot. in 2001, when no crystal structures were available for any of these proteins, anantharaman, koonin, and aravind identified homology between spou and trmd mtases and defined the spout superfamily. since then, multiple crystal structures of knotted mtases have been solved and numerous new homologous sequences appeared in the dat ...200717338813
temperature-dependent rnp conformational rearrangements: analysis of binary complexes of primary binding proteins with 16 s rrna.ribonucleoprotein particles (rnps) are important components of all living systems, and the assembly of these particles is an intricate, often multistep, process. the 30 s ribosomal subunit is composed of one large rna (16 s rrna) and 21 ribosomal proteins (r-proteins). in vitro studies have revealed that assembly of the 30 s subunit is a temperature-dependent process involving sequential binding of r-proteins and conformational changes of 16 s rrna. additionally, a temperature-dependent conforma ...200717376481
insertion sequence diversity in archaea.insertion sequences (iss) can constitute an important component of prokaryotic (bacterial and archaeal) genomes. over 1,500 individual iss are included at present in the isfinder database (www-is.biotoul.fr), and these represent only a small portion of those in the available prokaryotic genome sequences and those that are being discovered in ongoing sequencing projects. in spite of this diversity, the transposition mechanisms of only a few of these ubiquitous mobile genetic elements are known, a ...200717347521
intermediates: ubiquitous species on folding energy landscapes?although intermediates have long been recognised as fascinating species that form during the folding of large proteins, the role that intermediates play in the folding of small, single-domain proteins has been widely debated. recent discoveries using new, sensitive methods of detection and studies combining simulation and experiment have now converged on a common vision for folding, involving intermediates as ubiquitous stepping stones en route to the native state. the results suggest that the f ...200717239580
stabilization of alpha-chymotrypsin upon pegylation correlates with reduced structural dynamics.protein stability remains one of the main factors limiting the realization of the full potential of protein therapeutics. poly(ethylene glycol) (peg) conjugation to proteins has evolved into an important tool to overcome instability issues associated with proteins. the observed increase in thermodynamic stability of several proteins upon pegylation has been hypothesized to arise from reduced protein structural dynamics, although experimental evidence for this hypothesis is currently missing. to ...200818781698
how mitochondria produce reactive oxygen species.the production of ros (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. superoxide (o2(*-)) is the proximal mitochondrial ros, and in the present review i outline the principles that govern o2(*-) production within the matrix of mammalian mitochondria. the flux of o2(*-) is related to the concentration of potential electron donors, ...200819061483
how mitochondria produce reactive oxygen species.the production of ros (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. superoxide (o2(*-)) is the proximal mitochondrial ros, and in the present review i outline the principles that govern o2(*-) production within the matrix of mammalian mitochondria. the flux of o2(*-) is related to the concentration of potential electron donors, ...200819061483
upf201 archaeal specific family members reveal structural similarity to rna-binding proteins but low likelihood for rna-binding function.we have determined x-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (upf0201; pfam classification: duf54) to advance our understanding of the genetic repertoire of archaea. despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. their common polypeptide chain fold, encompassing a five-stranded antiparal ...200819079550
implementation of a flash-photolysis system for time-resolved cryo-electron microscopy.we describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. a previously designed computer-controlled cryo-plunging apparatus [white, h.d., thirumurugan, k., walker, m.l., trinick, j., 2003. a second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. j. struct. biol. 144, 246-252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. the irradiation in ...200819114106
implementation of a flash-photolysis system for time-resolved cryo-electron microscopy.we describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. a previously designed computer-controlled cryo-plunging apparatus [white, h.d., thirumurugan, k., walker, m.l., trinick, j., 2003. a second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. j. struct. biol. 144, 246-252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. the irradiation in ...200819114106
a bridge to transcription by rna polymerase.a comprehensive survey of single amino-acid substitution mutations critical for rna polymerase function published in journal of biology supports a proposed mechanism for polymerase action in which movement of the polymerase 'bridge helix' promotes transcriptional activity in cooperation with a critical substrate-interaction domain, the 'trigger loop'.200819090964
effect of primer proximity to a difficult-to-sequence region on read length and sequence quality.anecdotal and not well-established evidence implies that there could be some effect of primer proximity in relation to a difficult region on read length and sequence quality. in this paper we sequenced many different categories of difficult regions where primers were located at various distances in relation to such regions and we found that there is only weak, if any, correlation between primer proximity and read length or sequence quality. the occasional improvements observed in some studies co ...200819183797
the ua_handle: a versatile submotif in stable rna architectures.stable rnas are modular and hierarchical 3d architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the ua_handle motif (5'xu/an(n)x3'). it consists of a u:a watson-crick: hoogsteen trans base pair stacked over a classic watson-crick base pair, and a bulge of one or more nucleotides that can act as a handle for making ...200819036788
the ua_handle: a versatile submotif in stable rna architectures.stable rnas are modular and hierarchical 3d architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the ua_handle motif (5'xu/an(n)x3'). it consists of a u:a watson-crick: hoogsteen trans base pair stacked over a classic watson-crick base pair, and a bulge of one or more nucleotides that can act as a handle for making ...200819036788
genome-wide survey of prokaryotic serine proteases: analysis of distribution and domain architectures of five serine protease families in prokaryotes.serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. while studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. current analysis is aimed at understanding the distribution and probable b ...200819019219
the linkage between ribosomal crystallography, metal ions, heteropolytungstates and functional flexibility.crystallography of ribosomes, the universal cell nucleoprotein assemblies facilitating the translation of the genetic-code into proteins, met with severe problems owing to their large size, complex structure, inherent flexibility and high conformational variability. for the case of the small ribosomal subunit, which caused extreme difficulties, post crystallization treatment by minute amounts of a heteropolytungstate cluster allowed structure determination at atomic resolution. this cluster play ...200819915655
high tolerance for ionizable residues in the hydrophobic interior of proteins.internal ionizable groups are quite rare in water-soluble globular proteins. presumably, this reflects the incompatibility between charges and the hydrophobic environment in the protein interior. here we show that proteins can have an inherently high tolerance for internal ionizable groups. the 25 internal positions in staphylococcal nuclease were substituted one at a time with lys, glu, or asp without abolishing enzymatic activity and without detectable changes in the conformation of the protei ...200819004768
recognition of a common rdna target site in archaea and eukarya by analogous laglidadg and his-cys box homing endonucleases.the presence of a homing endonuclease gene (heg) within a microbial intron or intein empowers the entire element with the ability to invade genomic targets. the persistence of a homing endonuclease lineage depends in part on conservation of its dna target site. one such rdna sequence has been invaded both in archaea and in eukarya, by laglidadg and his-cys box homing endonucleases, respectively. the bases encoded by this target include a universally conserved ribosomal structure, termed helix 69 ...200818984620
molecular characterization of organelle-type nudix hydrolases in arabidopsis.nudix (for nucleoside diphosphates linked to some moiety x) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. arabidopsis (arabidopsis thaliana) contains 27 genes encoding nudix hydrolase homologues (atnudx1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. previously, cytosolic nudix hydrolases (atnudx1 to -11 and -25) were characterized. here, we conducted a charact ...200818815383
rna in motion.although rna duplex regions are highly structured and inflexible, other elements of an rna molecule are capable of dynamic motions. these flexible regions are the sites of interactions with small molecules, proteins, and other rnas, yet there are few descriptions of these regions that include the timescale and amplitude of their motions. no one technique is sufficient to accurately describe these motions, but the combination of in vitro methods, particularly nmr relaxation methods, and more robu ...200818957331
higher-order association states of cellular erbb3 probed with photo-cross-linkable aptamers.nucleic acid aptamers are rapidly gaining prominence as diagnostic tools, targeting reagents, and potential therapeutics. to extend the use of aptamers into the biochemical analysis of protein interactions on the surface of live cells, we converted an enzymatically generated rna aptamer into a photo-cross-linkable affinity tag through the replacement of all uracils with 4-thiouracil. specifically, we converted a previously selected, inhibitory aptamer that binds the soluble extracellular domains ...200818942860
structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch.the tetracycline aptamer is an in vitro selected rna that binds to the antibiotic with the highest known affinity of an artificial rna for a small molecule (kd approximately 0.8 nm). it is one of few aptamers known to be capable of modulating gene expression in vivo. the 2.2 a resolution cocrystal structure of the aptamer reveals a pseudoknot-like fold formed by tertiary interactions between an 11 nucleotide loop and the minor groove of an irregular helix. tetracycline binds within this interfac ...200818940672
rna folding and ribosome assembly.ribosome synthesis is a tightly regulated process that is crucial for cell survival. chemical footprinting, mass spectrometry, and cryo-electron microscopy are revealing how these complex cellular machines are assembled. rapid folding of the rrna provides a platform for protein-induced assembly of the bacterial 30s ribosome. multiple assembly pathways increase the flexibility of the assembly process, while accessory factors and modification enzymes chaperone the late stages of assembly and contr ...200818935976
dna polymerases and aminoacyl-trna synthetases: shared mechanisms for ensuring the fidelity of gene expression.dna polymerases and aminoacyl-trna synthetases (arss) represent large enzyme families with critical roles in the transformation of genetic information from dna to rna to protein. dna polymerases carry out replication and collaborate in the repair of the genome, while arss provide aminoacylated trna precursors for protein synthesis. enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high le ...200818850722
conformationally gated metal uptake by apomanganese superoxide dismutase.metal uptake by apomanganese superoxide dismutase in vitro is a complex process exhibiting multiphase "gated" reaction kinetics and a striking sigmoidal temperature profile that has led to a model of conformationally gated metal binding, requiring conversion between "closed" and "open" forms. this work systematically explores the structural determinants of metal binding in both wild-type (wt) apoprotein and mutational variants as a test of mechanistic models. the ph dependence of metalation unde ...200818841998
the higher level of organization of the oxidative phosphorylation system: mitochondrial supercomplexes.the organization of the oxidative phosphorylation (oxphos) system within the inner mitochondrial membrane appears to be far more complicated than previously thought. in particular, the individual protein complexes of the oxphos system (complexes i to v) were found to specifically interact forming defined supramolecular structures. blue-native polyacrylamide gel electrophoresis and single particle electron microscopy proved to be especially valuable in studying the so-called "respiratory supercom ...200818839290
the pretranslocation ribosome is targeted by gtp-bound ef-g in partially activated form.translocation of the trna x mrna complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor g (ef-g). we have used isothermal titration calorimetry to characterize the binding of gdp and gtp to free ef-g at 4 degrees c, 20 degrees c, and 37 degrees c. the binding affinity of ef-g is higher to gdp than to gtp at 4 degrees c, but lower at 37 degrees c. the binding enthalpy and entropy change little with temperature in the case of gdp binding but ...200818836081
the unique nature of mg2+ channels.considering the biological abundance and importance of mg2+, there is a surprising lack of information regarding the proteins that transport mg2+, the mechanisms by which they do so, and their physiological roles within the cell. the best characterized mg2+ channel to date is the bacterial protein cora, present in a wide range of bacterial species. the cora homolog mrs2 forms the mitochondrial mg2+ channel in all eukaryotes. physiologically, cora is involved in bacterial pathogenesis, and the mr ...200818927203
the metabolism of proline as microenvironmental stress substrate.proline, a unique proteogenic secondary amino acid, has its own metabolic system with special features. recent findings defining the regulation of this system led us to propose that proline is a stress substrate in the microenvironment of inflammation and tumorigenesis. the criteria for proline as a stress substrate are: 1) the enzymes utilizing proline respond to stress signaling; 2) there is a large, mobilizable pool of proline; and 3) the metabolism of proline serves special stress functions. ...200818806116
the medium-chain dehydrogenase/reductase engineering database: a systematic analysis of a diverse protein family to understand sequence-structure-function relationship.the medium-chain dehydrogenase/reductase engineering database (mdred, http://www.mdred.uni-stuttgart.de) has been established to serve as an analysis tool for a systematic investigation of sequence-structure-function relationships. it includes sequence and structure information of 2684 and 42 medium-chain dehydrogenases/reductases (mdrs), respectively. although mdrs are very diverse in sequence, they have a conserved tertiary structure. mdrs are assigned to 199 homologous families and 29 superfa ...200818614751
visualization of the eef2-80s ribosome transition-state complex by cryo-electron microscopy.in an attempt to understand ribosome-induced gtp hydrolysis on eef2, we determined a 12.6-a cryo-electron microscopy reconstruction of the eef2-bound 80s ribosome in the presence of aluminum tetrafluoride and gdp, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. this is the first visualization of a structure representing a transition-state complex on the ribosome. tight interactions are observed between the factor's g domain and the large ribosomal subunit, as well as ...200818644383
effects of protein subunits removal on the computed motions of partial 30s structures of the ribosome.the anisotropic network model (anm) is used to study motions of the 30s small ribosomal subunit. the effect of the absence of certain subunits on the motions of the remaining partial structures was investigated by removing one protein, pairs of proteins and selected sets of proteins at a time. our results show that the removal of some proteins doesn't change the large-scale dynamics of the partial structures, but the removal of certain subunits does cause significant changes in motion of the rem ...200819771145
genome signature analysis of thermal virus metagenomes reveals archaea and thermophilic signatures.metagenomic analysis provides a rich source of biological information for otherwise intractable viral communities. however, study of viral metagenomes has been hampered by its nearly complete reliance on blast algorithms for identification of dna sequences. we sought to develop algorithms for examination of viral metagenomes to identify the origin of sequences independent of blast algorithms. we chose viral metagenomes obtained from two hot springs, bear paw and octopus, in yellowstone national ...200818798991
evolution of catalases from bacteria to humans.excessive hydrogen peroxide is harmful for almost all cell components, so its rapid and efficient removal is of essential importance for aerobically living organisms. conversely, hydrogen peroxide acts as a second messenger in signal-transduction pathways. h(2)o(2) is degraded by peroxidases and catalases, the latter being able both to reduce h(2)o(2) to water and to oxidize it to molecular oxygen. nature has evolved three protein families that are able to catalyze this dismutation at reasonable ...200818498226
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
fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes.the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ...200818160515
fitness of streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase iv recombinant genes.the low prevalence of ciprofloxacin-resistant (cp r) streptococcus pneumoniae isolates carrying recombinant topoisomerase iv genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal pare-parc intergenic regions. a study of the transcription of these genes and of the fitness cost for 24 isogenic cp r strains was performed. six first-level transformants were obtained either with pcr products containing the parc quinol ...200818160515
early steps in the dna base excision/single-strand interruption repair pathway in mammalian cells.base excision repair (ber) is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged (oxidized or alkylated) or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species (ros). ber involves 4-5 steps starting with base excision by a dna glycosylase, followed by a common pathway usually involving an ap-endonuclease (ape) to generate 3' oh terminus at the damage site, followed by repai ...200818166975
kinetic pathway of pyrophosphorolysis by a retrotransposon reverse transcriptase.dna and rna polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. we previously showed, for the reverse transcriptase (rt) encoded by the yeast retrotransposon ty1, that one of the three conserved active site aspartates (d(211)) can be substituted by asparagine and still retain in vitro polymerase activity, although in vivo transposition is lost. transposition is partially restored by second site supp ...200818167548
structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine.the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ...200818171624
structures of open (r) and close (t) states of prephenate dehydratase (pdt)--implication of allosteric regulation by l-phenylalanine.the enzyme prephenate dehydratase (pdt) converts prephenate to phenylpyruvate in l-phenylalanine biosynthesis. pdt is allosterically regulated by l-phe and other amino acids. we report the first crystal structures of pdt from staphylococcus aureus in a relaxed (r) state and pdt from chlorobium tepidum in a tense (t) state. the two enzymes show low sequence identity (27.3%) but the same prototypic architecture and domain organization. both enzymes are tetramers (dimer of dimers) in crystal and so ...200818171624
identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna.the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ...200818177894
identification and role of functionally important motifs in the 970 loop of escherichia coli 16s ribosomal rna.the 970 loop (helix 31) of escherichia coli 16s ribosomal rna contains two modified nucleotides, m(2)g966 and m(5)c967. positions a964, a969, and c970 are conserved among the bacteria, archaea, and eukarya. the nucleotides present at positions 965, 966, 967, 968, and 971, however, are only conserved and unique within each domain. all organisms contain a modified nucleoside at position 966, but the type of the modification is domain specific. biochemical and structure studies have placed this loo ...200818177894
structural biophysics of the nusb:nuse antitermination complex.in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ...200818177898
structural biophysics of the nusb:nuse antitermination complex.in prokaryotic transcription regulation, several host factors form a complex with rna polymerase and the nascent mrna. as part of a process known as antitermination, two of these host factors, nusb and nuse, bind to form a heterodimer, which interacts with a specific boxa site on the rna. the nusb/nuse/boxa rna ternary complex interacts with the rna polymerase transcription complex, stabilizing it and allowing transcription past premature termination points. the nusb protein also binds boxa rna ...200818177898
structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase.aedes aegypti kynurenine aminotransferase (aekat) is a multifunctional aminotransferase. it catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. aekat also is a cysteine s-conjugate beta-lyase. the most important function of aekat is the biosynthesis of kynurenic acid, a natural antagonist of nmda and alpha7-nicotinic acetylcholine receptors. here, we report the crystal structures of aekat in complex with its best ...200818186649
cross-crystal averaging reveals that the structure of the peptidyl-transferase center is the same in the 70s ribosome and the 50s subunit.recently, two crystal structures of the thermus thermophilus 70s ribosome in the same functional state were determined at 2.8 and 3.7 a resolution but were different throughout. the most functionally significant structural differences are in the conformation of the peptidyl-transferase center (ptc) and the interface between the ptc and the cca end of the p-site trna. likewise, the 3.7 a ptc differed from the functionally equivalent structure of the haloarcula marismortui 50s subunit. to ascertai ...200818187576
evolution of mal abc transporter operons in the thermococcales and thermotogales.the mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea thermococcus litoralis and pyrococcus furiosus. bacterial hyperthermophiles of the order thermotogales live among these archaea and so may have shared in these transfers. the genome sequence of thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. we examined deep phylogenetic relationships among the m ...200818197971
nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase.isocitrate dehydrogenase (icdh) from hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. in this study, the oxidation reaction catalyzed by h. thermophilus icdh was kinetically analyzed. as a result, a rapid equilibrium random-order mechanism was suggested. the affinities of both substrates (isocitrate and nad+) toward the enzyme were extremely low ...200818203822
solution structure of ribosomal protein l40e, a unique c4 zinc finger protein encoded by archaeon sulfolobus solfataricus.the ribosomal protein l40e from archaeon sulfolobus solfataricus is a component of the 50s ribosomal subunit. l40e is a 56-residue, highly basic protein that contains a c4 zinc finger motif, crkc_x(10)_crrc. homologs are found in both archaea and eukaryotes but are not present in bacteria. eukaryotic genomes encode l40e as a ubiquitin-fusion protein. l40e was absent from the crystal structure of euryarchaeota 50s ribosomal subunit. here we report the three-dimensional solution structure of l40e ...200818218710
the solution structure of ribosomal protein s17e from methanobacterium thermoautotrophicum: a structural homolog of the ff domain.the ribosomal protein s17e from the archaeon methanobacterium thermoautotrophicum is a component of the 30s ribosomal subunit. s17e is a 62-residue protein conserved in archaea and eukaryotes and has no counterparts in bacteria. mammalian s17e is a phosphoprotein component of eukaryotic ribosomes. archaeal s17e proteins range from 59 to 79 amino acids, and are about half the length of the eukaryotic homologs which have an additional c-terminal region. here we report the three-dimensional solutio ...200818218711
molecular and physiological role of the trehalose-hydrolyzing alpha-glucosidase from thermus thermophilus hb27.trehalose supports the growth of thermus thermophilus strain hb27, but the absence of obvious genes for the hydrolysis of this disaccharide in the genome led us to search for enzymes for such a purpose. we expressed a putative alpha-glucosidase gene (ttc0107), characterized the recombinant enzyme, and found that the preferred substrate was alpha,alpha-1,1-trehalose, a new feature among alpha-glucosidases. the enzyme could also hydrolyze the disaccharides kojibiose and sucrose (alpha-1,2 linkage) ...200818223075
novel monofunctional histidinol-phosphate phosphatase of the dddd superfamily of phosphohydrolases.the ton_0887 gene was identified as the missing histidinol-phosphate phosphatase (holpase) in the hyperthermophilic archaeon "thermococcus onnurineus" na1. the protein contained conserved motifs of the dddd superfamily of phosphohydrolase, and the recombinantly expressed protein exhibited strong holpase activity. in this study, we functionally assessed for the first time the monofunctional dddd-type holpase, which is organized in the gene cluster.200818223080
novel members of glycoside hydrolase family 13 derived from environmental dna.starch and pullulan-modifying enzymes of the alpha-amylase family (glycoside hydrolase family 13) have several industrial applications. to date, most of these enzymes have been derived from isolated organisms. to increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using dna from enrichments from geothermal habitats. with this approach, we succeeded in isolating three new enzymes: a neopullulana ...200818223106
evidence for the involvement of acid/base chemistry in the reaction catalyzed by the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase from staphylococcus aureus.the type ii isopentenyl diphosphate/dimethylallyl diphosphate isomerase (idi-2) is a flavin mononucleotide (fmn)-dependent enzyme that catalyzes the reversible isomerization of isopentenyl pyrophosphate (ipp) to dimethylallyl pyrophosphate (dmapp), a reaction with no net change in redox state of the coenzyme or substrate. here, uv-vis spectral analysis of the idi-2 reaction revealed the accumulation of a reduced neutral dihydroflavin intermediate when the reduced enzyme was incubated with ipp or ...200818229948
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