Publications

the k-turn motif in riboswitches and other rna species.	the kink turn is a widespread structure motif that introduces a tight bend into the axis of duplex rna. this generally functions to mediate tertiary interactions, and to serve as a specific protein binding site. k-turns or closely related structures are found in at least seven different riboswitch structures, where they function as key architectural elements that help generate the ligand binding pocket. this article is part of a special issue entitled: riboswitches.	2014	24798078
cas1-cas2 complex formation mediates spacer acquisition during crispr-cas adaptive immunity.	the initial stage of crispr-cas immunity involves the integration of foreign dna spacer segments into the host genomic crispr locus. the nucleases cas1 and cas2 are the only proteins conserved among all crispr-cas systems, yet the molecular functions of these proteins during immunity are unknown. here we show that cas1 and cas2 from escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-å-resolution crystal structure of the cas1-cas2 complex. mutati ...	2014	24793649
a pause sequence enriched at translation start sites drives transcription dynamics in vivo.	transcription by rna polymerase (rnap) is interrupted by pauses that play diverse regulatory roles. although individual pauses have been studied in vitro, the determinants of pauses in vivo and their distribution throughout the bacterial genome remain unknown. using nascent transcript sequencing, we identified a 16-nucleotide consensus pause sequence in escherichia coli that accounts for known regulatory pause sites as well as ~20,000 new in vivo pause sites. in vitro single-molecule and ensembl ...	2014	24789973
involvement of proline oxidase (puta) in programmed cell death of xanthomonas.	xanthomonas campestris strains have been reported to undergo programmed cell death (pcd) in a protein rich medium. protein hydrolysates used in media such as nutrient broth comprise of casein digest with abundance of proline and glutamate. in the current study, x. campestris pv. campestris (xcc) cells displayed pcd when grown in pcd inducing medium (pim) containing casein tryptic digest. this pcd was also observed in pcd non-inducing carbohydrate rich medium (pnim) fortified with either proline ...	2014	24788936
protein thermodynamics can be predicted directly from biological growth rates.	life on earth is capable of growing from temperatures well below freezing to above the boiling point of water, with some organisms preferring cooler and others hotter conditions. the growth rate of each organism ultimately depends on its intracellular chemical reactions. here we show that a thermodynamic model based on a single, rate-limiting, enzyme-catalysed reaction accurately describes population growth rates in 230 diverse strains of unicellular and multicellular organisms. collectively the ...	2014	24787650
analysis of seca dimerization in solution.	the sec pathway mediates translocation of protein across the inner membrane of bacteria. seca is a motor protein that drives translocation of preprotein through the secyeg channel. seca reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in seca crystal structures. here, we have used biophysical approaches to address the nature of the seca dimer that exists in solution. we have taken advantage of the extreme salt sensitivity of seca dimerization ...	2014	24786965
metal-free camp-dependent protein kinase can catalyze phosphoryl transfer.	x-ray structures of several ternary product complexes of the catalytic subunit of camp-dependent protein kinase (pkac) have been determined with no bound metal ions and with na(+) or k(+) coordinated at two metal-binding sites. the metal-free pkac and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. in all studied complexes, the atp and the substrate peptide (sp20) were modified into the products adp and the phosphorylated peptide. the products of the phosp ...	2014	24786636
biochemical aspects of bacterial strategies for handling the incomplete translation processes.	during protein synthesis in cells, translating ribosomes may encounter abnormal situations that lead to retention of immature peptidyl-trna on the ribosome due to failure of suitable termination processes. bacterial cells handle such situations by employing three systems that rescue the stalled translation machinery. the transfer messenger rna/small protein b (tmrna/smpb) system, also called the trans-translation system, rescues stalled ribosomes by initiating template switching from the incompl ...	2014	24782856
secondary structure preferences of mn (2+) binding sites in bacterial proteins.	3d structures of proteins with coordinated mn(2+) ions from bacteria with low, average, and high genomic gc-content have been analyzed (149 pdb files were used). major mn(2+) binders are aspartic acid (6.82% of asp residues), histidine (14.76% of his residues), and glutamic acid (3.51% of glu residues). we found out that the motif of secondary structure "beta strand-major binder-random coil" is overrepresented around all the three major mn(2+) binders. that motif may be followed by either alpha ...	2014	24778647
tmrna-mediated trans-translation as the major ribosome rescue system in a bacterial cell.	transfer messenger rna (tmrna; also known as 10sa rna or ssra rna) is a small rna molecule that is conserved among bacteria. it has structural and functional similarities to trna: it has an upper half of the trna-like structure, its 5' end is processed by rnase p, it has typical trna-specific base modifications, it is aminoacylated with alanine, it binds to ef-tu after aminoacylation and it enters the ribosome with ef-tu and gtp. however, tmrna lacks an anticodon, and instead it has a coding seq ...	2014	24778639
electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex i.	complex i serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. it catalyzes the reduction of quinones by electron transfer from nadh, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. the membrane domain of the enzyme extends ∼180 å from the site of quinone reduction to the most distant proton pathway. to elucidate possible mechanisms of the long-range proton-coupled electron transfer pro ...	2014	24778264
comparative structural modeling of six old yellow enzymes (oyes) from the necrotrophic fungus ascochyta rabiei: insight into novel oye classes with differences in cofactor binding, organization of active site residues and stereopreferences.	old yellow enzyme (oye1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. in a recent study, we systematically identified oye proteins from various fungi and classified them into three classes viz. class i, ii and iii. however, there is no information about the structural organization of class iii oyes, eukaryotic class ii oyes and class ...	2014	24776850
genome-scale metabolic network reconstruction and in silico flux analysis of the thermophilic bacterium thermus thermophilus hb27.	thermus thermophilus, an extremely thermophilic bacterium, has been widely recognized as a model organism for studying how microbes can survive and adapt under high temperature environment. however, the thermotolerant mechanisms and cellular metabolism still remains mostly unravelled. thus, it is highly required to consider systems biological approaches where t. thermophilus metabolic network model can be employed together with high throughput experimental data for elucidating its physiological ...	2014	24774833
classification of intrinsically disordered regions and proteins.		2014	24773235
a novel approach to simulate a charge transfer in dna repair by an anacystis nidulans photolyase.	an anacystis nidulans photolyase enzyme containing two chromophore cofactors was simulated for a photoreaction dna repairing process via molecular dynamics (md) method. a novel approach has been introduced for the electron transfer between the fad (flavin adenine dinucleotide; flavin) molecule and cpd (cyclobutane pyrimidine dimer). this approach involves four simulation stages with different charges for the fad and cpd fragments and a role of a charged state of the active cofactor was qualified ...	2014	24772194
rna epigenetics.	mammalian messenger rna (mrna) and long noncoding rna (lncrna) contain tens of thousands of posttranscriptional chemical modifications. among these, the n(6)-methyl-adenosine (m(6)a) modification is the most abundant and can be removed by specific mammalian enzymes. m(6)a modification is recognized by families of rna binding proteins that affect many aspects of mrna function. mrna/lncrna modification represents another layer of epigenetic regulation of gene expression, analogous to dna methylati ...	2014	24768686
rna epigenetics.	mammalian messenger rna (mrna) and long noncoding rna (lncrna) contain tens of thousands of posttranscriptional chemical modifications. among these, the n(6)-methyl-adenosine (m(6)a) modification is the most abundant and can be removed by specific mammalian enzymes. m(6)a modification is recognized by families of rna binding proteins that affect many aspects of mrna function. mrna/lncrna modification represents another layer of epigenetic regulation of gene expression, analogous to dna methylati ...	2014	24768686
biosynthesis and functions of sulfur modifications in trna.	sulfur is an essential element for a variety of cellular constituents in all living organisms. in trna molecules, there are many sulfur-containing nucleosides, such as the derivatives of 2-thiouridine (s(2)u), 4-thiouridine (s(4)u), 2-thiocytidine (s(2)c), and 2-methylthioadenosine (ms(2)a). earlier studies established the functions of these modifications for accurate and efficient translation, including proper recognition of the codons in mrna or stabilization of trna structure. in many cases, ...	2014	24765101
metalloproteins containing cytochrome, iron-sulfur, or copper redox centers.		2014	24758379
chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial trna(leu(uur)) and trna(lys).	5-taurinomethyluridine (τm(5)u) and 5-taurinomethyl-2-thiouridine (τm(5)s(2)u) are located at the wobble position of human mitochondrial (hmt) trna(leu(uur)) and trna(lys), respectively. both hypermodified units restrict decoding of the third codon letter to a and g. pathogenic mutations in the genes encoding hmt-trna(leu(uur)) and hmt-trna(lys) are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (melas, merrf ...	2014	24757169
structural, kinetic and proteomic characterization of acetyl phosphate-dependent bacterial protein acetylation.	the emerging view of nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (ptm) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. this ptm is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme a (accoa) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. here, we ...	2014	24756028
a new spanner in the works of bacterial transcription.	a promising molecular target that is unlikely to develop antibiotic resistance has been identified in bacteria.	2014	24755293
ge23077 binds to the rna polymerase 'i' and 'i+1' sites and prevents the binding of initiating nucleotides.	using a combination of genetic, biochemical, and structural approaches, we show that the cyclic-peptide antibiotic ge23077 (ge) binds directly to the bacterial rna polymerase (rnap) active-center 'i' and 'i+1' nucleotide binding sites, preventing the binding of initiating nucleotides, and thereby preventing transcription initiation. the target-based resistance spectrum for ge is unusually small, reflecting the fact that the ge binding site on rnap includes residues of the rnap active center that ...	2014	24755292
a role for [fe4s4] clusters in trna recognition--a theoretical study.	over the past several years, structural studies have led to the unexpected discovery of iron-sulfur clusters in enzymes that are involved in dna replication/repair and protein biosynthesis. although these clusters are generally well-studied cofactors, their significance in the new contexts often remains elusive. one fascinating example is a tryptophanyl-trna synthetase from the thermophilic bacterium thermotoga maritima, tmtrprs, that has recently been structurally characterized. it represents a ...	2014	24753428
cas6 specificity and crispr rna loading in a complex crispr-cas system.	crispr-cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. in type i and type iii crispr-cas systems, crispr rna (crrna) is generated by cleavage of a primary transcript by the cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. highly studied crispr-cas systems such as those in escherichia coli and pseudomonas aeruginosa have a sin ...	2014	24753403
amino acid-dependent stability of the acyl linkage in aminoacyl-trna.	aminoacyl-trnas are the biologically active substrates for peptide bond formation in protein synthesis. the stability of the acyl linkage in each aminoacyl-trna, formed through an ester bond that connects the amino acid carboxyl group with the trna terminal 3'-oh group, is thus important. while the ester linkage is the same for all aminoacyl-trnas, the stability of each is not well characterized, thus limiting insight into the fundamental process of peptide bond formation. here, we show, by anal ...	2014	24751649
casa mediates cas3-catalyzed target degradation during crispr rna-guided interference.	in bacteria, the clustered regularly interspaced short palindromic repeats (crispr)-associated (cas) dna-targeting complex cascade (crispr-associated complex for antiviral defense) uses crispr rna (crrna) guides to bind complementary dna targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (pam). the cascade complex then recruits cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded dna (dsdna) bearing a sequen ...	2014	24748111
from metagenomics to pure culture: isolation and characterization of the moderately halophilic bacterium spiribacter salinus gen. nov., sp. nov.	recent metagenomic studies on saltern ponds with intermediate salinities have determined that their microbial communities are dominated by both euryarchaeota and halophilic bacteria, with a gammaproteobacterium closely related to the genera alkalilimnicola and arhodomonas being one of the most predominant microorganisms, making up to 15% of the total prokaryotic population. here we used several strategies and culture media in order to isolate this organism in pure culture. we report the isolatio ...	2014	24747894
controlling protein adsorption on graphene for cryo-em using low-energy hydrogen plasmas.	despite its many favorable properties as a sample support for biological electron microscopy, graphene is not widely used because its hydrophobicity precludes reliable protein deposition. we describe a method to modify graphene with a low-energy hydrogen plasma, which reduces hydrophobicity without degrading the graphene lattice. use of plasma-treated graphene enables better control of protein distribution in ice for electron cryo-microscopy and improves image quality by reducing radiation-induc ...	2014	24747813
mass spectrometry defines the c-terminal dimerization domain and enables modeling of the structure of full-length ompa.	the transmembrane domain of the outer membrane protein a (ompa) from escherichia coli is an excellent model for structural and folding studies of β-barrel membrane proteins. however, full-length ompa resists crystallographic efforts, and the link between its function and tertiary structure remains controversial. here we use site-directed mutagenesis and mass spectrometry of different constructs of ompa, released in the gas phase from detergent micelles, to define the minimal region encompassing ...	2014	24746938
biochemical and functional characterization of plasmodium falciparum gtp cyclohydrolase i.	antifolates are currently in clinical use for malaria preventive therapy and treatment. the drugs kill the parasites by targeting the enzymes in the de novo folate pathway. the use of antifolates has now been limited by the spread of drug-resistant mutations. gtp cyclohydrolase i (gch1) is the first and the rate-limiting enzyme in the folate pathway. the amplification of the gch1 gene found in certain plasmodium falciparum isolates can cause antifolate resistance and influence the course of anti ...	2014	24745605
mechanism of oxidant-induced mistranslation by threonyl-trna synthetase.	aminoacyl-trna synthetases maintain the fidelity during protein synthesis by selective activation of cognate amino acids at the aminoacylation site and hydrolysis of misformed aminoacyl-trnas at the editing site. threonyl-trna synthetase (thrrs) misactivates serine and utilizes an editing site cysteine (c182 in escherichia coli) to hydrolyze ser-trna(thr). hydrogen peroxide oxidizes c182, leading to ser-trna(thr) production and mistranslation of threonine codons as serine. the mechanism of c182 ...	2014	24744241
functional substitution of a eukaryotic glycyl-trna synthetase with an evolutionarily unrelated bacterial cognate enzyme.	two oligomeric types of glycyl-trna synthetase (glyrs) are found in nature: a α2 type and a α2β2 type. the former has been identified in all three kingdoms of life and often pairs with trnagly that carries an a73 discriminator base, while the latter is found only in bacteria and chloroplasts and is almost always coupled with trnagly that contains u73. in the yeast saccharomyces cerevisiae, a single glyrs gene, grs1, provides both the cytoplasmic and mitochondrial functions, and trnagly isoaccept ...	2014	24743154
non-nearest-neighbor dependence of stability for group iii rna single nucleotide bulge loops.	thirty-five rna duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. the bulge loops were of the group iii variety, where the bulged nucleotide is either a ag/u or cu/g, leading to ambiguity to the exact position and identity of the bulge. all possible group iii bulge loops with watson-crick nearest-neighbors were examined. the data were used to develop a model to predict the free energy of an rna duplex containing a ...	2014	24742935
a technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads for x-ray diffraction experiments.	a simple technique for high-throughput protein crystallization in ionically cross-linked polysaccharide gel beads has been developed for contactless handling of crystals in x-ray crystallography. the method is designed to reduce mechanical damage to crystals caused by physical contact between crystal and mount tool and by osmotic shock during various manipulations including cryoprotection, heavy-atom derivatization, ligand soaking, and diffraction experiments. for this study, protein crystalliza ...	2014	24740192
a novel carbonyl reductase with anti-prelog stereospecificity from acetobacter sp. cctcc m209061: purification and characterization.	a novel carbonyl reductase (accr) catalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-prelog stereoselectivity was found in acetobacter sp. cctcc m209061 and enriched 27.5-fold with an overall yield of 0.4% by purification. the enzyme showed a homotetrameric structure with an apparent molecular mass of 104 kda and each subunit of 27 kda. the gene sequence of accr was cloned and sequenced, and a 762 bp gene fragment was obtained. either nad(h) or nadp(h) can be used a ...	2014	24740089
binding and channeling of alternative substrates in the enzyme dmpfg: a molecular dynamics study.	dmpfg is a bifunctional enzyme comprised of an aldolase subunit, dmpg, and a dehydrogenase subunit, dmpf. the aldehyde intermediate produced by the aldolase is channeled directly through a buried molecular channel in the protein structure from the aldolase to the dehydrogenase active site. in this study, we have investigated the binding of a series of progressively larger substrates to the aldolase, dmpg, using molecular dynamics. all substrates investigated are easily accommodated within the ac ...	2014	24739167
crystal structures of the structure-selective nuclease mus81-eme1 bound to flap dna substrates.	the mus81-eme1 complex is a structure-selective endonuclease with a critical role in the resolution of recombination intermediates during dna repair after interstrand cross-links, replication fork collapse, or double-strand breaks. to explain the molecular basis of 3' flap substrate recognition and cleavage mechanism by mus81-eme1, we determined crystal structures of human mus81-eme1 bound to various flap dna substrates. mus81-eme1 undergoes gross substrate-induced conformational changes that re ...	2014	24733841
diversity of the metal-transporting p1b-type atpases.	the p1b-atpases are integral membrane proteins that couple atp hydrolysis to metal cation transport. widely distributed across all domains of life, these enzymes have been previously shown to transport copper, zinc, cobalt, and other thiophilic heavy metals. recent data suggest that these enzymes may also be involved in nickel and/or iron transport. here we have exploited large amounts of genomic data to examine and classify the various p1b-atpase subfamilies. specifically, we have combined new ...	2014	24729073
molecular recognition and modification of the 30s ribosome by the aminoglycoside-resistance methyltransferase npma.	aminoglycosides are potent, broad spectrum, ribosome-targeting antibacterials whose clinical efficacy is seriously threatened by multiple resistance mechanisms. here, we report the structural basis for 30s recognition by the novel plasmid-mediated aminoglycoside-resistance rrna methyltransferase a (npma). these studies are supported by biochemical and functional assays that define the molecular features necessary for npma to catalyze m(1)a1408 modification and confer resistance. the requirement ...	2014	24717845
mycobacterial rna polymerase requires a u-tract at intrinsic terminators and is aided by nusg at suboptimal terminators.	intrinsic terminators, which encode gc-rich rna hairpins followed immediately by a 7-to-9-nucleotide (nt) u-rich "u-tract," play principal roles of punctuating and regulating transcription in most bacteria. however, canonical intrinsic terminators with strong u-tracts are underrepresented in some bacterial lineages, notably mycobacteria, leading to proposals that their rna polymerases stop at noncanonical intrinsic terminators encoding various rna structures lacking u-tracts. we generated recomb ...	2014	24713321
trans-translation exposed: understanding the structures and functions of tmrna-smpb.	ribosome stalling is a serious issue for cell survival. in bacteria, the primary rescue system is trans-translation, performed by tmrna and its protein partner small protein b (smpb). since its discovery almost 20 years ago, biochemical, genetic, and structural studies have paved the way to a better understanding of how this sophisticated process takes place at the cellular and molecular levels. here we describe the molecular details of trans-translation, with special mention of recent cryo-elec ...	2014	24711807
bacteriophage λ n protein inhibits transcription slippage by escherichia coli rna polymerase.	transcriptional slippage is a class of error in which ribonucleic acid (rna) polymerase incorporates nucleotides out of register, with respect to the deoxyribonucleic acid (dna) template. this phenomenon is involved in gene regulation mechanisms and in the development of diverse diseases. the bacteriophage λ n protein reduces transcriptional slippage within actively growing cells and in vitro. n appears to stabilize the rna/dna hybrid, particularly at the 5' end, preventing loss of register betw ...	2014	24711367
annotation of protein domains reveals remarkable conservation in the functional make up of proteomes across superkingdoms.	the functional repertoire of a cell is largely embodied in its proteome, the collection of proteins encoded in the genome of an organism. the molecular functions of proteins are the direct consequence of their structure and structure can be inferred from sequence using hidden markov models of structural recognition. here we analyze the functional annotation of protein domain structures in almost a thousand sequenced genomes, exploring the functional and structural diversity of proteomes. we find ...	2011	24710297
polyamines in response to abiotic stress tolerance through transgenic approaches.	the distribution, growth, development and productivity of crop plants are greatly affected by various abiotic stresses. worldwide, sustainable crop productivity is facing major challenges caused by abiotic stresses by reducing the potential yield in crop plants by as much as 70%. plants can generally adapt to one or more environmental stresses to some extent. physiological and molecular studies at transcriptional, translational, and transgenic plant levels have shown the pronounced involvement o ...	2014	24710064
characterization of the type 2 nadh:menaquinone oxidoreductases from staphylococcus aureus and the bactericidal action of phenothiazines.	methicillin-resistant staphylococcus aureus (mrsa) is currently one of the principal multiple drug resistant bacterial pathogens causing serious infections, many of which are life-threatening. consequently, new therapeutic targets are required to combat such infections. in the current work, we explore the type 2 nicotinamide adenine dinucleotide reduced form (nadh) dehydrogenases (ndh-2s) as possible drug targets and look at the effects of phenothiazines, known to inhibit ndh-2 from mycobacteriu ...	2014	24709059
capturing the bacterial holo-complex.		2014	24707040
the lyr protein subunit nb4m/ndufa6 of mitochondrial complex i anchors an acyl carrier protein and is essential for catalytic activity.	mitochondrial complex i is the largest and most complicated enzyme of the oxidative phosphorylation system. it comprises a number of so-called accessory subunits of largely unknown structure and function. here we studied subunit nb4m [ndufa6, lyr motif containing protein 6 (lyrm6)], a member of the lyrm family of proteins. chromosomal deletion of the corresponding gene in the yeast yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit acpm1 from the enzyme ...	2014	24706851
understanding the mechanism of proteasome 20s core particle gating.	the 20s core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. although atomic resolution models of both open and c ...	2014	24706783
trna synthetase: trna aminoacylation and beyond.	the aminoacyl-trna synthetases are prominently known for their classic function in the first step of protein synthesis, where they bear the responsibility of setting the genetic code. each enzyme is exquisitely adapted to covalently link a single standard amino acid to its cognate set of trna isoacceptors. these ancient enzymes have evolved idiosyncratically to host alternate activities that go far beyond their aminoacylation role and impact a wide range of other metabolic pathways and cell sign ...	2014	24706556
off-resonance rotating-frame relaxation dispersion experiment for 13c in aromatic side chains using l-optimized trosy-selection.	protein dynamics on the microsecond-millisecond time scales often play a critical role in biological function. nmr relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. to date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites ...	2014	24706175
design of activated serine-containing catalytic triads with atomic-level accuracy.	a challenge in the computational design of enzymes is that multiple properties, including substrate binding, transition state stabilization and product release, must be simultaneously optimized, and this has limited the absolute activity of successful designs. here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. we design proteins with idealized serine-containing catalytic triads and assess their nucleophilicity dir ...	2014	24705591
thermodynamic and kinetic insights into stop codon recognition by release factor 1.	stop codon recognition is a crucial event during translation termination and is performed by class i release factors (rf1 and rf2 in bacterial cells). recent crystal structures showed that stop codon recognition is achieved mainly through a network of hydrogen bonds and stacking interactions between the stop codon and conserved residues in domain ii of rf1/rf2. additionally, previous studies suggested that recognition of stop codons is coupled to proper positioning of rf1 on the ribosome, which ...	2014	24699820
the structures of the cuta1 proteins from thermus thermophilus and pyrococcus horikoshii: characterization of metal-binding sites and metal-induced assembly.	cuta1 (copper tolerance a1) is a widespread cytoplasmic protein found in archaea, bacteria, plants and animals, including humans. in escherichia coli it is implicated in divalent metal tolerance, while the mammalian cuta1 homologue has been proposed to mediate brain enzyme acetylcholinesterase activity and copper homeostasis. the x-ray structures of cuta1 from the thermophilic bacterium thermus thermophilus (ttcuta1) with and without bound na(+) at 1.7 and 1.9 å resolution, respectively, and fro ...	2014	24699729
insights into protein allostery in the csor/rcnr family of transcriptional repressors.	csor/rcnr transcriptional repressors adopt a disc-shaped, all α-helical dimer of dimers tetrameric architecture, with a four-helix bundle the key structural feature of the dimer. individual members of this large family of repressors coordinate cu(i) or ni(ii)/co(ii) or perform cysteine sulfur chemistry in mitigating the effects of metal or metabolite toxicity, respectively. here we highlight recent insights into the functional diversity of this fascinating family of repressors.	2013	24695963
insights into protein allostery in the csor/rcnr family of transcriptional repressors.	csor/rcnr transcriptional repressors adopt a disc-shaped, all α-helical dimer of dimers tetrameric architecture, with a four-helix bundle the key structural feature of the dimer. individual members of this large family of repressors coordinate cu(i) or ni(ii)/co(ii) or perform cysteine sulfur chemistry in mitigating the effects of metal or metabolite toxicity, respectively. here we highlight recent insights into the functional diversity of this fascinating family of repressors.	2013	24695963
evidence of positive selection in mitochondrial complexes i and v of the african elephant.	as species evolve, they become adapted to their local environments. detecting the genetic signature of selection and connecting that to the phenotype of the organism, however, is challenging. here we report using an integrative approach that combines dna sequencing with structural biology analyses to assess the effect of selection on residues in the mitochondrial dna of the two species of african elephants. we detected evidence of positive selection acting on residues in complexes i and v, and w ...	2014	24695069
crystal structure of saccharomyces cerevisiae mitochondrial gatfab reveals a novel subunit assembly in trna-dependent amidotransferases.	yeast mitochondrial gln-mtrnagln is synthesized by the transamidation of mischarged glu-mtrnagln by a non-canonical heterotrimeric trna-dependent amidotransferase (adt). the gata and gatb subunits of the yeast adt (gatfab) are well conserved among bacteria and eukaryota, but the gatf subunit is a fungi-specific ortholog of the gatc subunit found in all other known heterotrimeric adts (gatcab). here we report the crystal structure of yeast mitochondrial gatfab at 2.0 å resolution. the c-terminal ...	2014	24692665
randomly selected suppressor mutations in genes for nadh : quinone oxidoreductase-1, which rescue motility of a salmonella ubiquinone-biosynthesis mutant strain.	the primary mobile electron-carrier in the aerobic respiratory chain of salmonella is ubiquinone. demethylmenaquinone and menaquinone are alternative electron-carriers involved in anaerobic respiration. ubiquinone biosynthesis was disrupted in strains bearing deletions of the ubia or ubie genes. in soft tryptone agar both mutant strains swam poorly. however, the ubia deletion mutant strain produced suppressor mutant strains with somewhat rescued motility and growth. six independent suppressor mu ...	2014	24692644
helix capping in rna structure.	helices are an essential element in defining the three-dimensional architecture of structured rnas. while internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. while coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in rna crystal structures, herein we ...	2014	24691270
measuring the shapes of macromolecules - and why it matters.	the molecular basis of life rests on the activity of biological macromolecules, mostly nucleic acids and proteins. a perhaps surprising finding that crystallized over the last handful of decades is that geometric reasoning plays a major role in our attempt to understand these activities. in this paper, we address this connection between geometry and biology, focusing on methods for measuring and characterizing the shapes of macromolecules. we briefly review existing numerical and analytical appr ...	2013	24688748
directed evolution: selection of the host organism.	directed evolution has become a well-established tool for improving proteins and biological systems. a critical aspect of directed evolution is the selection of a suitable host organism for achieving functional expression of the target gene. to date, most directed evolution studies have used either escherichia coli or saccharomyces cerevisiae as a host; however, other bacterial and yeast species, as well as mammalian and insect cell lines, have also been successfully used. recent advances in syn ...	2012	24688653
biotechnology of polyketides: new breath of life for the novel antibiotic genetic pathways discovery through metagenomics.	the discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. in fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the "goose that laid the golden egg," the potential of this wealth is still inexorable: simply ad ...	2014	24688489
biotechnology of polyketides: new breath of life for the novel antibiotic genetic pathways discovery through metagenomics.	the discovery of secondary metabolites produced by microorganisms (e.g., penicillin in 1928) and the beginning of their industrial application (1940) opened new doors to what has been the main medication source for the treatment of infectious diseases and tumors. in fact, approximately 80 years after the discovery of the first antibiotic compound, and despite all of the warnings about the failure of the "goose that laid the golden egg," the potential of this wealth is still inexorable: simply ad ...	2014	24688489
superoxide dismutases and superoxide reductases.		2014	24684599
an in silico approach for characterization of an aminoglycoside antibiotic-resistant methyltransferase protein from pyrococcus furiosus (dsm 3638).	pyrococcus furiosus is a hyperthermophilic archaea. a hypothetical protein of this archaea, pf0847, was selected for computational analysis. basic local alignment search tool and multiple sequence alignment (msa) tool were employed to search for related proteins. both the secondary and tertiary structure prediction were obtained for further analysis. three-dimensional model was assessed by procheck and qmean6 programs. to get insights about the physical and functional associations of the protein ...	2014	24683305
rna-seq-based analysis of cold shock response in thermoanaerobacter tengcongensis, a bacterium harboring a single cold shock protein encoding gene.	although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. thermoanaerobacter tengcongensis mb4, a thermophile harboring only one known cold shock protein gene (ttescpc), can survive from 50° to 80 °c, but has poor natural competence under cold shock at 50 °c. we therefore examined cold shock responses ...	2014	24667527
metal specificity of cyanobacterial nickel-responsive repressor inrs: cells maintain zinc and copper below the detection threshold for inrs.	inrs is a ni(ii)-responsive, csor/rcnr-like, dna-binding transcriptional repressor of the nrsd gene, but the ni(ii) co-ordination sphere of inrs is unlike ni(ii)-rcnr. we show that copper and zn(ii) also bind tightly to inrs and in vitro these ions also impair inrs binding to the nrsd operator-promoter. inrs does not respond to zn(ii) (or copper) in vivo after 48 h, when zn(ii) sensor ziar responds, but inrs transiently responds (1 h) to both metals. inrs conserves only one (of two) second co-or ...	2014	24666373
identifying mrna sequence elements for target recognition by human argonaute proteins.	it is commonly known that mammalian micrornas (mirnas) guide the rna-induced silencing complex (risc) to target mrnas through the seed-pairing rule. however, recent experiments that coimmunoprecipitate the argonaute proteins (agos), the central catalytic component of risc, have consistently revealed extensive ago-associated mrnas that lack seed complementarity with mirnas. we herein test the hypothesis that ago has its own binding preference within target mrnas, independent of guide mirnas. by s ...	2014	24663241
molecular basis for erythromycin-dependent ribosome stalling during translation of the ermbl leader peptide.	in bacteria, ribosome stalling during translation of ermbl leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ermb. the lack of structures of drug-dependent stalled ribosome complexes (srcs) has limited our mechanistic understanding of this regulatory process. here we present a cryo-electron microscopy structure of the erythromycin-dependent ermbl-src. the structure reveals that the an ...	2014	24662426
mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.	tuberculosis remains a major health concern worldwide. eradication of its causative agent, the bacterial pathogen mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. hypoxic conditions have been widely used as an in vitro system to study this dormancy. such studies identified a r ...	2014	24659783
cation diffusion facilitators transport initiation and regulation is mediated by cation induced conformational changes of the cytoplasmic domain.	cation diffusion facilitators (cdf) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. cdf's were shown to be involved in several human diseases, such as type-ii diabetes and neurodegenerative diseases. in this work, we employed a multi-disciplinary approach to study the activation mechanism of the cdf protein family. for this we used mamm, one of the main ion transporters of magnetosomes--bacterial organelles that enable mag ...	2014	24658343
moyamoya disease-associated protein mysterin/rnf213 is a novel aaa+ atpase, which dynamically changes its oligomeric state.	moyamoya disease is an idiopathic human cerebrovascular disorder that is characterized by progressive stenosis and abnormal collateral vessels. we recently identified mysterin/rnf213 as its first susceptibility gene, which encodes a 591-kda protein containing enzymatically active p-loop atpase and ubiquitin ligase domains and is involved in proper vascular development in zebrafish. here we demonstrate that mysterin further contains two tandem aaa+ atpase modules and forms huge ring-shaped oligom ...	2014	24658080
origin and evolution of the peptidyl transferase center from proto-trnas.	we tested the hypothesis of tamura (2011) [3] that molecules of trna gave origin to ribosomes, particularly to the peptidyl transferase center (ptc) of the 23s ribosomal rna. we reconstructed the ancestral sequences from all types of trna and compared them in their sequences with the current ptc of 23s ribosomal rna from different organisms. we built an ancestral sequence of proto-trnas that showed a remarkable overall identity of 50.53% with the catalytic site of ptc. we conclude that the pepti ...	2014	24649398
a sco protein among the hypothetical proteins of bacillus lehensis g1: its 3d macromolecular structure and association with cytochrome c oxidase.	at least a quarter of any complete genome encodes for hypothetical proteins (hps) which are largely non-similar to other known, well-characterized proteins. predicting and solving their structures and functions is imperative to aid understanding of any given organism as a complete biological system. the present study highlights the primary effort to classify and cluster 1202 hps of bacillus lehensis g1 alkaliphile to serve as a platform to mine and select specific hp(s) to be studied further in ...	2014	24641837
structure of the carboxy-terminal domain of mycobacterium tuberculosis card protein: an essential rrna transcriptional regulator.	the card protein is highly expressed in mycobacterial strains under basal conditions and is transcriptionally induced during multiple types of genotoxic stress and starvation. the card protein binds the β subunit of rna polymerase and influences gene expression. the disruption of interactions between card and the β subunit of rna polymerase has a significant effect on mycobacterial survival, resistance to stress and pathogenesis. to understand the structure of card and its interaction with the β ...	2014	24637748
rna helicase proteins as chaperones and remodelers.	superfamily 2 helicase proteins are ubiquitous in rna biology and have an extraordinarily broad set of functional roles. central among these roles are the promotion of rearrangements of structured rnas and the remodeling of ribonucleoprotein complexes (rnps), allowing formation of native rna structure or progression through a functional cycle of structures. although all superfamily 2 helicases share a conserved helicase core, they are divided evolutionarily into several families, and it is princ ...	2014	24635478
pyrrolysyl-trna synthetase: an ordinary enzyme but an outstanding genetic code expansion tool.	the genetic incorporation of the 22nd proteinogenic amino acid, pyrrolysine (pyl) at amber codon is achieved by the action of pyrrolysyl-trna synthetase (pylrs) together with its cognate trna(pyl). unlike most aminoacyl-trna synthetases, pylrs displays high substrate side chain promiscuity, low selectivity toward its substrate α-amine, and low selectivity toward the anticodon of trna(pyl). these unique but ordinary features of pylrs as an aminoacyl-trna synthetase allow the pyl incorporation mac ...	2014	24631543
the substrate specificity, enantioselectivity and structure of the (r)-selective amine : pyruvate transaminase from nectria haematococca.	during the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (s)-enantiospecificity. transaminases with (r)-specificity are needed to expand the applications of these enzymes in biocatalysis. in this work we have identified a fungal putative (r)-specific transaminase from the eurotiomycetes nect ...	2014	24618038
chlamydia exploit the mammalian tryptophan-depletion defense strategy as a counter-defensive cue to trigger a survival state of persistence.	we previously proposed that in chlamydiaceae rapid vegetative growth and a quiescent state of survival (persistence) depend upon alternative protein translational profiles dictated by host tryptophan (trp) availability. these alternative profiles correspond, respectively, with a set of chlamydial proteins having higher-than-predicted contents of trp ("up-trp" selection), or with another set exhibiting lower-than-predicted contents of trp ("down-trp" selection). a comparative evaluation of chlamy ...	2014	24616884
identification of a novel aminopropyltransferase involved in the synthesis of branched-chain polyamines in hyperthermophiles.	longer- and/or branched-chain polyamines are unique polycations found in thermophiles. n(4)-aminopropylspermine is considered a major polyamine in thermococcus kodakarensis. to determine whether a quaternary branched penta-amine, n(4)-bis(aminopropyl)spermidine, an isomer of n(4)-aminopropylspermine, was also present, acid-extracted cytoplasmic polyamines were analyzed by high-pressure liquid chromatography, gas chromatography (hplc), and gas chromatography-mass spectrometry. n(4)-bis(aminopropy ...	2014	24610711
automated particle picking for low-contrast macromolecules in cryo-electron microscopy.	cryo-electron microscopy is an increasingly popular tool for studying the structure and dynamics of biological macromolecules at high resolution. a crucial step in automating single-particle reconstruction of a biological sample is the selection of particle images from a micrograph. we present a novel algorithm for selecting particle images in low-contrast conditions; it proves more effective than the human eye on close-to-focus micrographs, yielding improved or comparable resolution in reconstr ...	2014	24607413
adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes.	human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. nevertheless, the receptor for human resistin has not been clarified. here, we identified adenylyl cyclase-associated protein 1 (cap1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. we found that human r ...	2014	24606903
mechanism of trans-translation revealed by in vitro studies.	tmrna is a bacterial small rna having a structure resembling the upper half of trna and its 3' end accepts alanine followed by binding to ef-tu like trna. instead of lacking a lower half of the cloverleaf structure including the anticodon, tmrna has a short coding sequence for tag-peptide that serves as a target of cellular proteases. an elaborate coordination of two functions as trna and mrna facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from ...	2014	24600445
carbohydrate metabolism in archaea: current insights into unusual enzymes and pathways and their regulation.	the metabolism of archaea, the third domain of life, resembles in its complexity those of bacteria and lower eukarya. however, this metabolic complexity in archaea is accompanied by the absence of many "classical" pathways, particularly in central carbohydrate metabolism. instead, archaea are characterized by the presence of unique, modified variants of classical pathways such as the embden-meyerhof-parnas (emp) pathway and the entner-doudoroff (ed) pathway. the pentose phosphate pathway is only ...	2014	24600042
bacterial genome instability.	bacterial genomes are remarkably stable from one generation to the next but are plastic on an evolutionary time scale, substantially shaped by horizontal gene transfer, genome rearrangement, and the activities of mobile dna elements. this implies the existence of a delicate balance between the maintenance of genome stability and the tolerance of genome instability. in this review, we describe the specialized genetic elements and the endogenous processes that contribute to genome instability. we ...	2014	24600039
tetartohedral twinning in idi-2 from thermus thermophilus: crystallization under anaerobic conditions.	type-2 isopentenyl diphosphate isomerase (idi-2) is a key flavoprotein involved in the biosynthesis of isoprenoids. since fully reduced flavin mononucleotide (fmnh2) is needed for activity, it was decided to crystallize the enzyme under anaerobic conditions in order to understand how this reduced cofactor binds within the active site and interacts with the substrate isopentenyl diphosphate (ipp). in this study, the protein was expressed and purified under aerobic conditions and then reduced and ...	2014	24598924
cbr antimicrobials alter coupling between the bridge helix and the β subunit in rna polymerase.	bacterial rna polymerase (rnap) is a validated target for antibacterial drugs. cbr703 series antimicrobials allosterically inhibit transcription by binding to a conserved α helix (β' bridge helix, bh) that interconnects the two largest rnap subunits. here we show that disruption of the bh-β subunit contacts by amino-acid substitutions invariably results in accelerated catalysis, slowed-down forward translocation and insensitivity to regulatory pauses. cbr703 partially reverses these effects in c ...	2014	24598909
structural insights into dna repair by rnase t--an exonuclease processing 3' end of structured dna in repair pathways.	dna repair mechanisms are essential for preservation of genome integrity. however, it is not clear how dna are selected and processed at broken ends by exonucleases during repair pathways. here we show that the dnaq-like exonuclease rnase t is critical for escherichia coli resistance to various dna-damaging agents and uv radiation. rnase t specifically trims the 3' end of structured dna, including bulge, bubble, and y-structured dna, and it can work with endonuclease v to restore the deaminated ...	2014	24594808
structural basis for promoter specificity switching of rna polymerase by a phage factor.	transcription of dna to rna by dna-dependent rna polymerase (rnap) is the first step of gene expression and a major regulation point. bacteriophages hijack their host's transcription machinery and direct it to serve their needs. the gp39 protein encoded by thermus thermophilus phage p23-45 binds the host's rnap and inhibits transcription initiation from its major "-10/-35" class promoters. phage promoters belonging to the minor "extended -10" class are minimally inhibited. we report the crystal ...	2014	24589779
new extremophilic lipases and esterases from metagenomics.	lipolytic enzymes catalyze the hydrolysis of ester bonds in the presence of water. in media with low water content or in organic solvents, they can catalyze synthetic reactions such as esterification and transesterification. lipases and esterases, in particular those from extremophilic origin, are robust enzymes, functional under the harsh conditions of industrial processes owing to their inherent thermostability and resistance towards organic solvents, which combined with their high chemo-, reg ...	2014	24588890
structure and mechanism of soybean atp sulfurylase and the committed step in plant sulfur assimilation.	enzymes of the sulfur assimilation pathway are potential targets for improving nutrient content and environmental stress responses in plants. the committed step in this pathway is catalyzed by atp sulfurylase, which synthesizes adenosine 5'-phosphosulfate (aps) from sulfate and atp. to better understand the molecular basis of this energetically unfavorable reaction, the x-ray crystal structure of atp sulfurylase isoform 1 from soybean (glycine max atp sulfurylase) in complex with aps was determi ...	2014	24584934
nature and biosynthesis of galacto-oligosaccharides related to oligosaccharides in human breast milk.	human milk oligosaccharides (hmo) are prominent among the functional components of human breast milk. while hmo have potential applications in both infants and adults, this potential is limited by the difficulties in manufacturing these complex structures. consequently, functional alternatives such as galacto-oligosaccharides are under investigation, and nowadays, infant formulae are supplemented with galacto-oligosaccharides to mimic the biological effects of hmo. recently, approaches toward th ...	2014	24571717
characterisation of the active/de-active transition of mitochondrial complex i.	oxidation of nadh in the mitochondrial matrix of aerobic cells is catalysed by mitochondrial complex i. the regulation of this mitochondrial enzyme is not completely understood. an interesting characteristic of complex i from some organisms is the ability to adopt two distinct states: the so-called catalytically active (a) and the de-active, dormant state (d). the a-form in situ can undergo de-activation when the activity of the respiratory chain is limited (i.e. in the absence of oxygen). the m ...	2014	24569053
rigidity analysis of protein biological assemblies and periodic crystal structures.	we initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. the goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. we use experimental x-ray crystallography data from the protein data bank (pdb). the analysis relies on an effcient graph-based algorithm. computational experiments were performed using new protein rigidity analysis tools av ...	2013	24564201
nd3, nd1 and 39kda subunits are more exposed in the de-active form of bovine mitochondrial complex i.	an intriguing feature of mitochondrial complex i from several species is the so-called a/d transition, whereby the idle enzyme spontaneously converts from the active (a) form to the de-active (d) form. the a/d transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. we demonstrate for the first time that the a/d conformational change of complex i does not aff ...	2014	24560811
high-affinity gold nanoparticle pin to label and localize histidine-tagged protein in macromolecular assemblies.	there is significant demand for experimental approaches to aid protein localization in electron microscopy micrographs and ultimately in three-dimensional reconstructions of macromolecular assemblies. we report preparation and use of a reagent consisting of tris-nitrilotriacetic acid (tris-nta) conjugated with a monofunctional gold nanoparticle ((aunp)tris-nta) for site-specific, non-covalent labeling of protein termini fused to a histidine-tag (his-tag). multivalent binding of tris-nta to a his ...	2014	24560806
insights into the glycosylase search for damage from single-molecule fluorescence microscopy.	the first step of base excision repair utilizes glycosylase enzymes to find damage within a genome. a persistent question in the field of dna repair is how glycosylases interact with dna to specifically find and excise target damaged bases with high efficiency and specificity. ensemble studies have indicated that glycosylase enzymes rely upon both sliding and distributive modes of search, but ensemble methods are limited in their ability to directly observe these modes. here we review insights i ...	2014	24560296
ion mobility-mass spectrometry of a rotary atpase reveals atp-induced reduction in conformational flexibility.	rotary atpases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. using ion mobility mass spectrometry we compared the conformational dynamics of the intact atpase from thermus thermophilus with those of its membrane and soluble subcomplexes. our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. to provide a structural context for ...	2014	24557135
paclitaxel induces apoptosis in breast cancer cells through different calcium--regulating mechanisms depending on external calcium conditions.	previously, we reported that endoplasmic reticulum calcium stores were a direct target for paclitaxel initiation of apoptosis. furthermore, the actions of paclitaxel attenuated bcl-2 resistance to apoptosis through endoplasmic reticulum-mediated calcium release. to better understand the calcium-regulated mechanisms of paclitaxel-induced apoptosis in breast cancer cells, we investigated the role of extracellular calcium, specifically; whether influx of extracellular calcium contributed to and/or ...	2014	24549172