Thermophilic Aquifex Aeolicus Research Articles

Structure–function analysis of tRNA t6A-catalysis, assembly and thermostability of Aquifex aeolicus TsaD2B2 tetramer in complex with TsaE

The universal N6-threonylcarbamoyladenosine (t6A) at position 37 of tRNAs is one of core post-transcriptional modifications that are needed for promoting translational fidelity. In bacteria, TsaC utilizes L-threonine, bicarbonate and ATP to generate an intermediate threonylcarbamoyladenylate (TC-AMP), of which the TC-moiety is transferred to N6 atom of tRNA A37 to generate t6A by TsaD with support of TsaB and TsaE. TsaD and TsaB form a TsaDB dimer to which tRNA and TsaE are competitively bound. The catalytic mechanism of TsaD and auxiliary roles of TsaB and TsaE remain to be fully elucidated. In this study, we reconstituted tRNA t6A biosynthesis using recombinant TsaC, TsaD–TsaB and TsaE from thermophilic Aquifex aeolicus and determined crystal structures of apo-form and ADP-bound form of TsaD2B2 tetramer. Our TsaD2B2–TsaE–tRNA model coupled functional validations reveal that the binding of tRNA or TsaE to TsaDB is regulated by C-terminal tail of TsaB and a helical hairpin α1–α2 of TsaD. A. aeolicus TsaD2B2 or TsaDB possesses a basal divalent ion-dependent t6A-catalytic activity that is stimulated by TsaE at the cost of ATP consumption. Our data suggest that binding of TsaE to TsaDB induces conformational changes of α1, α2, α6, α7 and α8 of TsaD and C-terminal tail of TsaB, leading to release of tRNA t6A and AMP. ATP hydrolysis-driven dissociation of TsaE from TsaDB resets an active conformation of TsaDB. Dimerization of thermophilic TsaDB enhances thermostability and promotes t6A-catalytic activity of TsaD2B2–tRNA, of which GC base pairs in anticodon stem are needed for correct folding of thermophilic tRNA at higher temperatures.

Design and Preclinical Evaluation of a Nanoparticle Vaccine against Respiratory Syncytial Virus Based on the Attachment Protein G.

Human respiratory syncytial virus (RSV) poses a significant human health threat, particularly to infants and the elderly. While efficacious vaccines based on the F protein have recently received market authorization, uncertainties remain regarding the future need for vaccine updates to counteract potential viral drift. The attachment protein G has long been ignored as a vaccine target due to perceived non-essentiality and ineffective neutralization on immortalized cells. Here, we show strong G-based neutralization in fully differentiated human airway epithelial cell (hAEC) cultures that is comparable to F-based neutralization. Next, we designed an RSV vaccine component based on the central conserved domain (CCD) of G fused to self-assembling lumazine synthase (LS) nanoparticles from the thermophile Aquifex aeolicus as a multivalent antigen presentation scaffold. These nanoparticles, characterized by high particle expression and assembly through the introduction of N-linked glycans, showed exceptional thermal and storage stability and elicited potent RSV neutralizing antibodies in a mouse model. In conclusion, our results emphasize the pivotal role of RSV G in the viral lifecycle and culminate in a promising next-generation RSV vaccine candidate characterized by excellent manufacturability and immunogenic properties. This candidate could function independently or synergistically with current F-based vaccines.

Voltage vs. Ligand II: Structural insights of the intrinsic flexibility in cyclic nucleotide-gated channels

ABSTRACTIn the preceding article, we present a flexibility analysis of the voltage-gated ion channel (VGIC) superfamily. In this study, we describe in detail the flexibility profile of the voltage-sensor domain (VSD) and the pore domain (PD) concerning the evolution of 6TM ion channels. In particular, we highlight the role of flexibility in the emergence of CNG channels and describe a significant level of sequence similarity between the archetypical VSD and the TolQ proteins. A highly flexible S4-like segment exhibiting Lys instead Arg for these membrane proteins is reported. Sequence analysis indicates that, in addition to this S4-like segment, TolQ proteins also show similarity with specific motifs in S2 and S3 from typical V-sensors. Notably, S3 flexibility profiles from typical VSDs and S3-like in TolQ proteins are also similar. Interestingly, TolQ from early divergent prokaryotes are comparatively more flexible than those in modern counterparts or true V-sensors. Regarding the PD, we also found that 2TM K+-channels in early prokaryotes are considerably more flexible than the ones in modern microbes, and such flexibility is comparable to the one present in CNG channels. Voltage dependence is mainly exhibited in prokaryotic CNG channels whose VSD is rigid whereas the eukaryotic CNG channels are considerably more flexible and poorly V-dependent. The implication of the flexibility present in CNG channels, their sensitivity to cyclic nucleotides and the cation selectivity are discussed. Finally, we generated a structural model of the putative cyclic nucleotide-modulated ion channel, which we coined here as AqK, from the thermophilic bacteria Aquifex aeolicus, one of the earliest diverging prokaryotes known. Overall, our analysis suggests that V-sensors in CNG-like channels were essentially rigid in early prokaryotes but raises the possibility that this module was probably part of a very flexible stator protein of the bacterial flagellum motor complex.

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

The bacterial flagellar motor is a self-assembling supramolecular nanodevice. Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the N-terminal domain of FliG (FliF C -FliG N ) from the bacterium Helicobacter pylori Within this complex, FliF C interacted with FliG N through extensive hydrophobic contacts similar to those observed in the FliF C -FliG N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF C -FliG N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo repeat-like motifs (ARM) of FliG N Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG N stacking by complementation studies in Escherichia coli Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG N , containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: conservation of the lateral RNA-binding mode.

The host factor Hfq, as the bacterial branch of the Sm family, is an RNA-binding protein involved in the post-transcriptional regulation of mRNA expression and turnover. Hfq facilitates pairing between small regulatory RNAs (sRNAs) and their corresponding mRNA targets by binding both RNAs and bringing them into close proximity. Hfq homologs self-assemble into homo-hexameric rings with at least two distinct surfaces that bind RNA. Recently, another binding site, dubbed the `lateral rim', has been implicated in sRNA·mRNA annealing; the RNA-binding properties of this site appear to be rather subtle, and its degree of evolutionary conservation is unknown. An Hfq homolog has been identified in the phylogenetically deep-branching thermophile Aquifex aeolicus (Aae), but little is known about the structure and function of Hfq from basal bacterial lineages such as the Aquificae. Therefore, Aae Hfq was cloned, overexpressed, purified, crystallized and biochemically characterized. Structures of Aae Hfq were determined in space groups P1 and P6, both to 1.5 Å resolution, and nanomolar-scale binding affinities for uridine- and adenosine-rich RNAs were discovered. Co-crystallization with U6 RNA reveals that the outer rim of the Aae Hfq hexamer features a well defined binding pocket that is selective for uracil. This Aae Hfq structure, combined with biochemical and biophysical characterization of the homolog, reveals deep evolutionary conservation of the lateral RNA-binding mode, and lays a foundation for further studies of Hfq-associated RNA biology in ancient bacterial phyla.

Fishing with flies, worms and bacteria: emerging models for mammalian membrane transport and trafficking

The Journal of Physiology has a long and distinguished history of publishing original observations using non-mammalian tractable model systems that have fundamentally informed understanding of mammalian membrane transport processes. Some specific examples include a pioneering investigation of the selective barrier function of epithelia using frog skin (Waymouth Reid, 1890), an elegant illustration of ion channel and Na+–K+ pump activity in giant axons from the cuttlefish and squid (Hodgkin & Keynes, 1955) and an early demonstration of Na+-dependent sugar transport in the toad intestine (Csaky & Thale, 1960). Such physiological demonstrations of transport function have, in more recent times, been supported by the explosion in available genomic information that has, in turn, greatly improved our understanding of conserved biological functions across many species. Likewise, the high degree of similarity in protein structures from prokaryotes and eukaryotes, despite limited sequence homology, also points towards essential and sustained biological roles. Thus, prokaryotic transporter structures can provide key information concerning the way in which mammalian transport proteins are likely to function. A series of talks on the topic of emerging models for investigation of mammalian membrane transport and trafficking were presented on Sunday 21 April 2013 as part of the American Physiological Society programme of symposia at the Experimental Biology 2013 meeting. The session was held in the Boston Convention & Exhibition Center. The symposium was sponsored by The Physiological Society and The Journal of Physiology. The topics covered in this symposium emphasized the utility of so-called ‘lower organisms’ and how they can be used to improve our understanding of mammalian membrane transport and trafficking. The symposium opened with a presentation by Aravind Penmatsa on how the elucidation of the crystal structure of the amino acid transporter LeuT, from the thermophilic bacterium Aquifex aeolicus, has revolutionized our understanding of mammalian neurotransmitter transporter stucture and function (Penmatsa & Gouaux, 2014). The availability of the LeuT crystal has provided a structural basis to determine the action of antidepressants on the SLC6 family of human biogenic amine transporters (Wang et al. 2013). The next speaker, Britta Spanier, highlighted clear similarities in the mechanisms of transcriptional and functional regulation of the intestinal di/tripeptide transporter PepT1 in the gut of the nematode Caenorhabditis elegans and the commonly used cell culture model of the human small intestinal epithelium, the Caco-2 cell monolayer (Spanier, 2014). In both cases, PepT1 is regulated by the intracellular amino acid pool and functional activity of the co-expressed Na+–H+ exchanger NHE3. Tiziano Verri continued the theme of intestinal absorption of protein by providing a comprehensive review of the genomic and functional information currently available about PepT1 from a great number of studies using teleost fish (Romano et al. 2014). Intestinal protein absorption is such a fundamental biological process that modifications of the PepT1 transporter protein in different teleosts allow adaptation to extremes of temperature, pH and substrate availability. This information not only provides useful tips on how the mammalian PepT1 could be modified to enhance transport but also provides valuable information for optimization of feed for commercially valuable fish. Linda Stuart gave an elegant presentation on how animals (ranging from the fruitfly Drosophila melanogaster to mammals) sense pathogens (Stuart et al. 2013) and how phagosome acidification following phagocytosis of Gram-positive and Gram-negative bacteria takes place via distinct mechanisms (Sokolovska et al. 2013). The session was completed by a short presentation by Bill Harvey on a novel theory that considers how a K+ pump, comprising a H+ V-ATPase and a K+–H+ antiporter, originally discovered in the caterpillar midgut, might contribute to auditory mechanosensory transduction in the mammalian cochlea (Harvey & Xiang, 2012). The topics discussed in this symposium where diverse, but the common thread is clear. Our understanding of mammalian physiology is enhanced greatly by information obtained from studies of diverse organisms, including prokaryotes, fish, flies and worms.

Structure, function, and tethering of DNA-binding domains in σ⁵⁴ transcriptional activators.

We compare the structure, activity, and linkage of DNA-binding domains (DBDs) from σ(54) transcriptional activators and discuss how the properties of the DBDs and the linker to the neighboring domain are affected by the overall properties and requirements of the full proteins. These transcriptional activators bind upstream of specific promoters that utilize σ(54)-polymerase. Upon receiving a signal the activators assemble into hexamers, which then, through adenosine triphosphate (ATP) hydrolysis, drive a conformational change in polymerase that enables transcription initiation. We present structures of the DBDs of activators nitrogen regulatory protein C 1 (NtrC1) and Nif-like homolog 2 (Nlh2) from the thermophile Aquifex aeolicus. The structures of these domains and their relationship to other parts of the activators are discussed. These structures are compared with previously determined structures of the DBDs of NtrC4, NtrC, ZraR, and factor for inversion stimulation. The N-terminal linkers that connect the DBDs to the central domains in NtrC1 and Nlh2 were studied and found to be unstructured. Additionally, a crystal structure of full-length NtrC1 was solved, but density of the DBDs was extremely weak, further indicating that the linker between ATPase and DBDs functions as a flexible tether. Flexible linking of ATPase and DBDs is likely necessary to allow assembly of the active hexameric ATPase ring. The comparison of this set of activators also shows clearly that strong dimerization of the DBD only occurs when other domains do not dimerize strongly.

FlgM proteins from different bacteria exhibit different structural characteristics

Intrinsically disordered proteins (IDPs) are a unique class of proteins that do not require a stable structure for function. The importance of IDPs in many biological processes has been established but there remain unanswered questions about their evolution and conservation of their disordered state within a protein family. Our group has been studying the structural similarities among orthologous FlgM proteins, a model class of IDPs. We have previously shown that the FlgM protein from the thermophile Aquifex aeolicus has more structure at A. aeolicus' physiological temperature (85°C) than is observed for the Salmonella typhimurium FlgM, suggesting that the disordered nature of FlgM varies among organisms and is not universally conserved. In this work, we extend these studies to the FlgM proteins from Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, and Bacillus subtilis. We demonstrate that the B. subtilis, E. coli, and S. typhimurium FlgMs exist in a premolten globule-like conformation, though the B. subtilis FlgM is in a more compacted conformation than the other two. The P. aeruginosa and P. mirabilis FlgM proteins exist in a currently unknown conformation that is not either coil-like or premolten globule-like. The P. aeruginosa FlgM appears to contain more weak intramolecular contacts given its more compacted state than the P. mirabilis FlgM. These results provide experimental evidence that members of the same protein family can exhibit different degrees of disorder, though understanding how different disordered states evolve in the same protein family will require more study.

3P200 高度好熱菌Aquifex aeolicus由来のべん毛モーター固定子タンパク質の性質検討(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

3P200 高度好熱菌Aquifex aeolicus由来のべん毛モーター固定子タンパク質の性質検討(12.細胞生物的課題,ポスター,日本生物物理学会年会第51回(2013年度))

Discrimination of Thermophilic and Mesophilic Proteins Using Reduced Amino Acid Alphabets with n-Grams

Protein thermostabilization has been the focus of recent research due to growing interest in the production of enzymes that can operate at temperatures that are industrially beneficial. Understanding the determinants of thermostabilization at the level of sequence and structure is important to design such enzymes. A bioinformatical approach was used to determine the extent by which reduced amino acid alphabets (RAAA) with n-grams (subsequences of length n) that were subjected to a t-test-based feature selection procedure can be used to discriminate proteins from thermophiles and mesophiles. Classification performance of 65 different protein alphabets with 3 different n-gram sizes was systematically evaluated using support vector machines in a test set that contained 707 proteins from mesophilic Xylella fastidosa and thermophilic Aquifex aeolicus. A classification accuracy of 91.796% was achieved with Hsdm16 RAAA with 13 features: EK-ILV-ST-A-G-F-H-Q-N-R-M-W-Y. The t-test-based feature selection procedure reduced the classification time without significantly affecting classification accuracy. The overall combination of methods in this paper is useful and computationally fast for classifying protein sequences from thermophiles and mesophiles using sequence information alone.

Crystal structure of prephenate dehydrogenase from Streptococcus mutans

Prephenate dehydrogenase (PDH) is a bacterial enzyme that catalyzes conversion of prephenate to 4-hydroxyphenylpyruvate through the oxidative decarboxylation pathway for tyrosine biosynthesis. This enzymatic pathway exists in prokaryotes but is absent in mammals, indicating that it is a potential target for the development of new antibiotics. The crystal structure of PDH from Streptococcus mutans in a complex with NAD+ shows that the enzyme exists as a homo-dimer, each monomer consisting of two domains, a modified nucleotide binding N-terminal domain and a helical prephenate C-terminal binding domain. The latter is the dimerization domain. A structural comparison of PDHs from mesophilic S. mutans and thermophilic Aquifex aeolicus showed differences in the long loop between β6 and β7, which may be a reason for the high Km values of PDH from Streptococcus mutans.

Aquifex aeolicus FlgM protein exhibits a temperature-dependent disordered nature

Studies on the nature and function of intrinsically disordered proteins (IDP) over the past 10 years have demonstrated the importance of IDPs in normal cellular function. Although many proteins predicted to be IDPs have been experimentally characterized on an individual basis, the conservation of disorder between homologous proteins from different organisms has not been fully studied. We now demonstrate that the FlgM protein from the thermophile Aquifex aeolicus exhibits a more ordered conformation at 20 degrees C than the previously characterized FlgM protein from Salmonella typhimurium. FlgM is an inhibitor of the RNA transcription factor sigma28, which is involved in regulation of the late-stage genes involved in flagella synthesis. Previous work has shown that the S. typhimurium FlgM protein is an intrinsically disordered protein, though the C-terminus becomes ordered when bound to sigma28 or under crowded solution conditions. In this work, we demonstrate that at 20 degrees C the A. aeolicus FlgM protein exhibits alpha-helical character in circular dichroism (CD) experiments, though the percentage of alpha-helical content decreases with increased temperature, consistent with the FlgM assuming a less folded conformation. We also show that the A. aeolicus FlgM exhibits cooperativity in chemical denaturation experiments, consistent with a globular nature. Furthermore, we use the fluorescent probe FlAsH to show that the H2 helix is ordered, even in the unbound state and that the H1 and H2 helices appear to be associated with each other in the absence of the sigma28 protein. Finally, we demonstrate that the H2 helix assumes an extended conformation at 85 degrees C. Based on our results, we propose that at 20 degrees C the A. aeolicus FlgM assumes a four-helix bundle-like conformation that becomes a more extended conformation at the A. aeolicus' physiological temperature of 85 degrees C.

Aquifex Aeolicus FlgM Protein Does Not Exhibit the Disordered Character of the Salmonella Typhimurium FlgM Protein

Studies on the nature and function of Intrinsically Disordered Proteins (IDP) over the past ten years have demonstrated the importance of IDPs in normal cellular function. The flexibility of IDPs allows one IDP to assume multiple conformations or form different protein-protein complexes, allowing a single protein to exhibit multiple functions. While many predicted IDPs have been characterized on an individual basis, the conservation of disorder between homologous proteins from different organisms has not been carefully studied. We now demonstrate that the FlgM protein from the thermophile Aquifex aeolicus exhibits significantly less disorder then the previously characterized FlgM protein from Salmonella typhimurium. FlgM is an inhibitor of the RNA transcription factor σ28, which is involved in regulation of flagella synthesis gene expression. Previous work has shown that the S. typhimurium FlgM protein is an intrinsically disordered protein, though the C-terminus becomes ordered when bound to σ28 or under crowded solution conditions. In this work, we demonstrate that, even under dilute solution conditions, that the A. aeolicus FlgM protein exhibits alpha-helical character. Furthermore, we use the fluorescent probe FlAsH to show that the H2 helix is ordered, even in the unbound state, in contrast to the S. typhimurium FlgM protein, and the H1 and H2 helices appear to be associated in the absence of the σ28 protein. Taken together, our data demonstrates that the A. aeolicus FlgM protein, while flexible, does not exhibit the intrinsically disordered nature exhibited by the S. typhimurium FlgM protein.

Hyperthermophilic Aquifex aeolicus initiates primer synthesis on a limited set of trinucleotides comprised of cytosines and guanines.

The placement of the extreme thermophile Aquifex aeolicus in the bacterial phylogenetic tree has evoked much controversy. We investigated whether adaptations for growth at high temperatures would alter a key functional component of the replication machinery, specifically DnaG primase. Although the structure of bacterial primases is conserved, the trinucleotide initiation specificity for A. aeolicus was hypothesized to differ from other microbes as an adaptation to a geothermal milieu. To determine the full range of A. aeolicus primase activity, two oligonucleotides were designed that comprised all potential trinucleotide initiation sequences. One of the screening templates supported primer synthesis and the lengths of the resulting primers were used to predict possible initiation trinucleotides. Use of trinucleotide-specific templates demonstrated that the preferred initiation trinucleotide sequence for A. aeolicus primase was 5′-d(CCC)-3′. Two other sequences, 5′-d(GCC)-3′ and d(CGC)-3′, were also capable of supporting initiation, but to a much lesser degree. None of these trinucleotides were known to be recognition sequences used by other microbial primases. These results suggest that the initiation specificity of A. aeolicus primase may represent an adaptation to a thermophilic environment.

Investigation on the Causes of Codon and Amino Acid Usages Variation Between Thermophilic Aquifex aeolicus and Mesophilic Bacillus subtilis

Base composition, codon usages and amino acid usages have been analyzed by taking 529 orthologous sequences of Aquifex aeolicus and Bacillus subtilis, having different optimal growth temperatures. These two bacteria do not have significant difference in overall GC composition, but GC(1+2) and GC3 levels were found to vary significantly. Significant increments in purine content and GC3 composition have been observed in the coding sequences of Aquifex aeolicus than its Bacillus subtilis counterparts. Correspondence analyses on codon and amino acid usages reveal that variation in base composition actually influences their codon and amino acid usages. Two selection pressures acting on the nucleotide level (GC3 and purine enrichment), causes variation in the amino acid usage differently in different protein secondary structures. Our results suggest that adaptation of amino acid usages in coil structure of Aquifex aeolicus proteins is under the control of both purine increment and GC3 composition, whereas the adaptation of the amino acids in the helical region of thermophilic bacteria is strongly influenced by the purine content. Evolutionary perspectives concerning the temperature adaptation of DNA and protein molecules of these two bacteria have been discussed on the basis of these results.

Regulation of the transcriptional activator NtrC1: structural studies of the regulatory and AAA+ ATPase domains.

Transcription by sigma54 RNA polymerase depends on activators that contain ATPase domains of the AAA+ class. These activators, which are often response regulators of two-component signal transduction systems, remodel the polymerase so that it can form open complexes at promoters. Here, we report the first crystal structures of the ATPase domain of an activator, the NtrC1 protein from the extreme thermophile Aquifex aeolicus. This domain alone, which is active, crystallized as a ring-shaped heptamer. The protein carrying both the ATPase and adjacent receiver domains, which is inactive, crystallized as a dimer. In the inactive dimer, one residue needed for catalysis is far from the active site, and extensive contacts among the domains prevent oligomerization of the ATPase domain. Oligomerization, which completes the active site, depends on surfaces that are buried in the dimer, and hence, on a rearrangement of the receiver domains upon phosphorylation. A motif in the ATPase domain known to be critical for coupling energy to remodeling of polymerase forms a novel loop that projects from the middle of an alpha helix. The extended, structured loops from the subunits of the heptamer localize to a pore in the center of the ring and form a surface that could contact sigma54.

Laccases and their occurrence in prokaryotes.

Laccases are copper-containing proteins that require O(2) to oxidize phenols, polyphenols, aromatic amines, and different non-phenolic substrates by one-electron transfer, resulting in the formation of reactive radicals. Although their specific physiological functions are not completely understood, there are several indications that laccases are involved in the morphogenesis of microorganisms (e.g., fungal spore development, melanization) and in the formation and/or degradation of complex organic substances such as lignin or humic matter. Owing to their high relative non-specific oxidation capacity, laccases are useful biocatalysts for diverse biotechnological applications. To date, laccases have been found only in eukaryotes (fungi, plants); however, databank searches and experimental data now provide evidence for their distribution in prokaryotes. This survey shows that laccase-like enzymes occur in many gram-negative and gram-positive bacteria. Corresponding genes have been found in prokaryotes that are thought to have branched off early during evolution, e.g., the extremely thermophilic Aquifex aeolicus and the archaeon Pyrobaculum aerophilum. Phylogenetically, the enzymes are members of the multi-copper protein family that have developed from small-sized prokaryotic azurins to eukaryotic plasma proteins.

The [formula omitted] subunit of Aquifex aeolicus leucyl-tRNA synthetase is responsible for cognate tRNA recognition

The active form of the leucyl-tRNA synthetase from an extreme thermophile Aquifex aeolicus has a heterodimeric (α/β type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases. In an attempt to clarify the individual roles of each subunit in the function of leucyl-tRNA synthetase, several elementary activities were separately measured using each of the subunits alone or the reconstructed α/β complex. It was found that the β subunit alone is capable of recognizing its cognate tRNA, while the leucyl-adenylate formation and the overall leucyl-tRNA formation are detected only when both of the subunit proteins coexisted.

Expression, refolding and crystallization ofAquifex aeolicuselongation factor P

Elongation factor P is a universally conserved protein stimulating peptidyltransferase activity during protein synthesis. The factor is sensitive to classical inhibitors of the ribosomal peptidyltransferase activity and is possibly involved in alignment of the substrate tRNAs in the catalytic centre of 70S ribosomes. Elongation factor P from the thermophilic Aquifex aeolicus was overexpressed as a soluble protein in Escherichia coli and crystallized. A fast generally applicable refolding protocol was developed to improve crystal quality and circumvent strong binding of oligonucleotides to the protein. Diffraction data collected to 2.7 A resolution present twinning.

Leucyl-tRNA synthetase from the extreme thermophile Aquifex aeolicus has a heterodimeric quaternary structure

Class I aminoacyl-tRNA synthetases have been thought to be single polypeptide enzymes. However, the complete genome sequence of a hyper thermophile Aquifex aeolicus suggests that the gene for leucyl-tRNA synthetases (LeuRS) is probably split into two pieces ( leuS and leuS′). In this research, each gene was separately cloned and overexpressed in Escherichia coli and the protein products were examined for LeuRS activity. Leucylation activity was detected only when both gene products coexisted. Gel filtration analysis showed that the active form of A. aeolicus LeuRS has a heterodimeric (α/β type) quaternary structure that is unique among class I aminoacyl-tRNA synthetases.