Role of the C-Terminal ß Sandwich of Thermoanaerobacter tengcongensis Thermophilic Esterase in Hydrolysis of Long-Chain Acyl Substrates.

To search for a novel thermostable esterase for optimized industrial applications, esterase from a thermophilic eubacterium species, Thermoanaerobacter tengcongensis MB4, was purified and characterized in this work. Sequence analysis of T. tengcongensis esterase with other homologous esterases of the same family revealed an apparent tail at the C-terminal that is not conserved across the esterase family. Hence, it was hypothesized that the tail is unlikely to have an essential structural or catalytic role. However, there is no documented report of any role for this tail region. We probed the role of the C-terminal domain on the catalytic activity and substrate preference of T. tengcongensis esterase EstA3 with a view to see how it could be engineered for enhanced properties. To achieve this, we cloned, expressed, and purified the wild-type and the truncated versions of the enzyme. In addition, a naturally occurring member of the family (from Brevibacillus brevis) that lacks the C-terminal tail was also made. In vitro characterization of the purified enzymes showed that the C-terminal domain contributes significantly to the catalytic activity and distinct substrate preference of T. tengcongensis esterase EstA3. All three recombinant enzymes showed the highest preference for paranitrophenyl butyrate (pNPC4), which suggests they are true esterases, not lipases. Kinetic data revealed that truncation had a slight effect on the substrate-binding affinity. Thus, the drop in preference towards long-chain substrates might not be a result of substrate binding affinity alone. The findings from this work could form the basis for future protein engineering allowing the modification of esterase catalytic properties through domain swapping or by attaching a modular protein domain.

Characterization of ferredoxins from the thermophilic, acetogenic bacterium Thermoanaerobacter kivui.

A major electron carrier involved in energy and carbon metabolism in the acetogenic model organism Thermoanaerobacter kivui is ferredoxin, an iron-sulfur-containing, electron-transferring protein. Here, we show that the genome of T. kivui encodes four putative ferredoxin-like proteins (TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530). All four genes were cloned, a His-tag encoding sequence was added and the proteins were produced from a plasmid in T. kivui. The purified proteins had an absorption peak at 430 nm typical for ferredoxins. The determined iron-sulfur content is consistent with the presence of two predicted [4Fe4S] clusters in TKV_c09620 and TKV_c19530 or one predicted [4Fe4S] cluster in TKV_c16450 and TKV_c10420 respectively. The reduction potential (Em ) for TKV_c09620, TKV_c16450, TKV_c10420 and TKV_c19530 was determined to be -386 ± 4 mV, -386 ± 2 mV, -559 ± 10 mV and -557 ± 3 mV, respectively. TKV_c09620 and TKV_c16450 served as electron carriers for different oxidoreductases from T. kivui. Deletion of the ferredoxin genes led to only a slight reduction of growth on pyruvate or autotrophically on H2 + CO2 . Transcriptional analysis revealed that TKV_c09620 was upregulated in a ΔTKV_c16450 mutant and vice versa TKV_c16450 in a ΔTKV_c09620 mutant, indicating that TKV_c09620 and TKV_c16450 can replace each other. In sum, our data are consistent with the hypothesis that TKV_c09620 and TKV_c16450 are ferredoxins involved in autotrophic and heterotrophic metabolism of T. kivui.

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium.

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (∆Tm -19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.

Synthesis of zinc-gallate phosphors by biomineralization and their emission properties.

Reduction of target species by microorganisms and their subsequent precipitation into sparingly soluble mineral phase nanoparticles have been referred to as microbially mediated nanomaterial synthesis. Here, we describe the microbially mediated production of nano-dimensioned spinel structured zinc-gallate (ZnGa2O4) phosphors exhibiting different emission performance with varying substituted elements. Interestingly, in the microbially mediated phosphor production described herein, there were no reducible metal- and non-metal species composing the target minerals. By varying substituted elements, zinc-gallate phosphors present typical red, green, and blue (RGB) emission. An apparent whitish emission was accomplished by blending phosphors. A promising potential for white light produced by biosynthesized mixtures of Cr-, Mn-, and Co- substituted zinc-gallates representing RGB emissions was evidenced. Microbial activity supplied a reducing driving force and provided appropriate near neutral pH and reduced Eh conditions to thermodynamically precipitate spinel structured nanomaterials from supersaturated divalent and trivalent cations. This result complemented conventional biomineralization concepts and expanded the realm of biomanufacturing nanomaterials for further applications. STATEMENT OF SIGNIFICANCE: This study substantiated that circumstances of a suitable pH/Eh derived from bacterial activity, divalent/trivalent ion supply, buffering capacity, and supersaturation could precipitate spinel structure nanoparticles. Even though live or dead cells with membrane could enhance the nuclei generation, the spinel structured phases were produced regardless of existence of live or dead cells and reducible metal or non-metal species incorporating into the produced solid phases. This finding led to production of a series of metal-substituted zinc-gallates with specific RGB emission that can result in whitish light using simple blending. We believe our findings could expand the realm of nanomaterial synthesis using low cost, highly scalable bio-nanotechnology.

Enzymatic Synthesis of a Novel Pterostilbene α-Glucoside by the Combination of Cyclodextrin Glucanotransferase and Amyloglucosidase.

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% (v/v) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae (var. diastaticus). This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

Automated NMR resonance assignments and structure determination using a minimal set of 4D spectra.

Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6-10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.

Small Molecule-Induced Domain Swapping as a Mechanism for Controlling Protein Function and Assembly.

Domain swapping is the process by which identical proteins exchange reciprocal segments to generate dimers. Here we introduce induced domain swapping (INDOS) as a mechanism for regulating protein function. INDOS employs a modular design consisting of the fusion of two proteins: a recognition protein that binds a triggering molecule, and a target protein that undergoes a domain swap in response to binding of the triggering ligand. The recognition protein (FK506 binding protein) is inserted into functionally-inactivated point mutants of two target proteins (staphylococcal nuclease and ribose binding protein). Binding of FK506 to the FKBP domain causes the target domain to first unfold, then refold via domain swap. The inactivating mutations become 'swapped out' in the dimer, increasing nuclease and ribose binding activities by 100-fold and 15-fold, respectively, restoring them to near wild-type values. INDOS is intended to convert an arbitrary protein into a functional switch, and is the first example of rational design in which a small molecule is used to trigger protein domain swapping and subsequent activation of biological function.

Conformational Rearrangements of Individual Nucleotides during RNA-Ligand Binding Are Rate-Differentiated.

A pronounced rate differentiation has been found for conformational rearrangements of individual nucleobases that occur during ligand recognition of the preQ1 class-I riboswitch aptamer from Thermoanaerobacter tengcongensis. Rate measurements rely on the 2ApFold approach by analyzing the fluorescence response of riboswitch variants, each with a single, strategically positioned 2-aminopurine nucleobase substitution. Observed rate discrimination between the fastest and the slowest conformational adaption is 22-fold, with the largest rate observed for the rearrangement of a nucleoside directly at the binding site and the smallest rate observed for the 3'-unpaired nucleoside that stacks onto the pseudo-knot-closing Watson-Crick base pair. Our findings provide novel insights into how compact, prefolded RNAs that follow the induced-fit recognition mechanism adapt local structural elements in response to ligand binding on a rather broad time scale and how this process culminates in a structural signal that is responsible for efficient downregulation of ribosomal translation.

Characterization of Electrical Current-Generation Capabilities from Thermophilic Bacterium Thermoanaerobacter pseudethanolicus Using Xylose, Glucose, Cellobiose, or Acetate with Fixed Anode Potentials.

Thermoanaerobacter pseudethanolicus 39E (ATCC 33223), a thermophilic, Fe(III)-reducing, and fermentative bacterium, was evaluated for its ability to produce current from four electron donors-xylose, glucose, cellobiose, and acetate-with a fixed anode potential (+ 0.042 V vs SHE) in a microbial electrochemical cell (MXC). Under thermophilic conditions (60 °C), T. pseudethanolicus produced high current densities from xylose (5.8 ± 2.4 A m(-2)), glucose (4.3 ± 1.9 A m(-2)), and cellobiose (5.2 ± 1.6 A m(-2)). It produced insignificant current when grown with acetate, but consumed the acetate produced from sugar fermentation to produce electrical current. Low-scan cyclic voltammetry (LSCV) revealed a sigmoidal response with a midpoint potential of -0.17 V vs SHE. Coulombic efficiency (CE) varied by electron donor, with xylose at 34.8% ± 0.7%, glucose at 65.3% ± 1.0%, and cellobiose at 27.7% ± 1.5%. Anode respiration was sustained over a pH range of 5.4-8.3, with higher current densities observed at higher pH values. Scanning electron microscopy showed a well-developed biofilm of T. pseudethanolicus on the anode, and confocal laser scanning microscopy demonstrated a maximum biofilm thickness (Lf) greater than ~150 µm for the glucose-fed biofilm.

Positive enrichment of C-terminal peptides using oxazolone chemistry and biotinylation.

Selective capture of protein C-termini is still challenging in view of the lower reactivity of the carboxyl group relative to amino groups and difficulties in site-specifically labeling the carboxyl group on the C-terminus rather than that on the side chains of acidic amino acids. For highly efficient purification of C-terminus peptides, a novel positive enrichment approach based on the oxazolone chemistry has been developed in this study. A bifunctional group reagent containing biotin and arginine was incorporated into the C-terminus of protein. Together with a streptavidin affinity strategy, the C-terminal peptides could be readily purified and analyzed by mass spectrometry (MS). Unlike the negative enrichment approach, C-terminal peptides, other than non-C-terminal peptides, were captured directly from the peptide mixture in this new method. The labeling efficiency (higher than 90%), enrichment selectivity (purifying C-terminal peptides from mixtures of non-C-terminal peptides at a 1:50 molar ratio), and ionization efficiencies in MS were dramatically improved. Moreover, the highly efficient identification of C-terminal peptides was further achieved by defining biotin as the 21st amino acid and optimizing the database search strategy. We have successfully identified 183 C-terminal peptides from Thermoanaerobacter tengcongensis using this creative method, which affords a highly selective and efficient purification approach for C-terminomics study.

Structures of Large RNAs and RNA-Protein Complexes: Toward Structure Determination of Riboswitches.

Riboswitches are widespread and important regulatory elements. They are typically present in the mRNA of the gene under their regulation, where they form complex three-dimensional structures that can bind an effector and regulate either transcription or translation of the mRNA. Structural biology has been essential to our understanding of their ligand recognition and conformational switching mechanisms, but riboswitch determination presents several important complications. Overcoming these challenges requires a synergistic approach using rational design of the constructs and supporting methods to biochemically validate the designs and resulting structures.

Structural and functional characterization of Cys4 zinc finger motif in the recombination mediator protein RecR.

Zinc finger motif widely exists in protein structure, which can play different roles in different proteins. RecR is an important recombination mediator protein (RMP) in the RecFOR pathway and zinc finger motif is the most conserved domain in RecR protein. However, the function of this zinc finger motif in RecR is unclear. Here, we have studied the structures of the single cysteine and double cysteines mutation within the zinc finger motif in Thermoanaerobacter tengcongensis RecR (TTERecR). We have also studied the DNA binding ability as well as TTERecO protein binding ability of single, double and even triple cysteines mutation of the zinc finger motif, and the mutants do not alter DNA binding by RecR nor the interaction between RecR and RecO. The function of TTERecR zinc finger motif is to maintain the stability of the three-dimensional structure.

Scalable production of microbially mediated zinc sulfide nanoparticles and application to functional thin films.

A series of semiconducting zinc sulfide (ZnS) nanoparticles were scalably, reproducibly, controllably and economically synthesized with anaerobic metal-reducing Thermoanaerobacter species. These bacteria reduced partially oxidized sulfur sources to sulfides that extracellularly and thermodynamically incorporated with zinc ions to produce sparingly soluble ZnS nanoparticles with ∼5nm crystallites at yields of ∼5gl(-1)month(-1). A predominant sphalerite formation was facilitated by rapid precipitation kinetics, a low cation/anion ratio and a higher zinc concentration compared to background to produce a naturally occurring hexagonal form at the low temperature, and/or water adsorption in aqueous conditions. The sphalerite ZnS nanoparticles exhibited narrow size distribution, high emission intensity and few native defects. Scale-up and emission tunability using copper doping were confirmed spectroscopically. Surface characterization was determined using Fourier transform infrared and X-ray photoelectron spectroscopies, which confirmed amino acid as proteins and bacterial fermentation end products not only maintaining a nano-dimensional average crystallite size, but also increasing aggregation. The application of ZnS nanoparticle ink to a functional thin film was successfully tested for potential future applications.

Thermal stability of Thermoanaerobacter tengcongensis ribosome recycling factor.

Ribosome recycling factor (RRF) and elongation factor-G catalyze disassembly of the post-termination complex and recycling of the ribosomal subunits back to a new round of initiation. Thermoanaerobacter tengcongensis survive high temperatures that Escherichia coli cannot, partly due to the higher thermal stability of T. tengcongensis ribosome recycling factor (tteRRF). Here we compared the structural stability of tteRRF and E. coli RRF (ecoRRF) and explore the reasons for the differences. We obtained the values of the thermodynamic parameters. Salt could reduce the thermal stability of tteRRF, which suggested that ion pairing was an important stabilizing factor in the case of tteRRF. The value of the heat capacity change of tteRRF unfolding, ΔCp, is significantly smaller than that of ecoRRF. A consequence of the small ΔCp value is that the change in free energy upon unfolding (ΔG) of tteRRF is larger than that of ecoRRF, while the values of the enthalpy change (ΔH) and the entropy change multiplied by temperature (T*ΔS) are smaller. The small ΔCp of tteRRF appears to be the main stabilizing factor for tteRRF.

Computational modeling of protein-RNA complex structures.

Protein-RNA interactions play fundamental roles in many biological processes, such as regulation of gene expression, RNA splicing, and protein synthesis. The understanding of these processes improves as new structures of protein-RNA complexes are solved and the molecular details of interactions analyzed. However, experimental determination of protein-RNA complex structures by high-resolution methods is tedious and difficult. Therefore, studies on protein-RNA recognition and complex formation present major technical challenges for macromolecular structural biology. Alternatively, protein-RNA interactions can be predicted by computational methods. Although less accurate than experimental measurements, theoretical models of macromolecular structures can be sufficiently accurate to prompt functional hypotheses and guide e.g. identification of important amino acid or nucleotide residues. In this article we present an overview of strategies and methods for computational modeling of protein-RNA complexes, including software developed in our laboratory, and illustrate it with practical examples of structural predictions.

Continuous production of ß-cyclodextrin from starch by highly stable cyclodextrin glycosyltransferase immobilized on chitosan.

Cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. was covalently immobilized on glutaraldehyde-activated chitosan spheres and used in a packed bed reactor to investigate the continuous production of ß-cyclodextrin (ß-CD). The optimum temperatures were 75 °C and 85 °C at pH 6.0, respectively for free and immobilized CGTase, and the optimum pH (5.0) was the same for both at 60 °C. In the reactor, the effects of flow rate and substrate concentration in the ß-CD production were evaluated. The optimum substrate concentration was 4% (w/v), maximizing the ß-CD production (1.32 g/L) in a flow rate of 3 mL/min. In addition, the biocatalyst had good operational stability at 60 °C, maintaining 61% of its initial activity after 100 cycles of batch and 100% after 100 h of continuous use. These results suggest the possibility of using this immobilized biocatalyst in continuous production of CDs.

Selectivity mechanism of the mechanosensitive channel MscS revealed by probing channel subconducting states.

The mechanosensitive channel of small conductance (MscS) has been characterized at both functional and structural levels and has an integral role in the protection of bacterial cells against hypoosmotic shock. Here we investigate the role that the cytoplasmic domain has in MscS channel function by recording wild-type and mutant MscS single-channel activity in liposome patches. We report that MscS preferentially resides in subconducting states at hyperpolarising potentials when Ca(2+) and Ba(2+) ions are the major permeant cations. In addition, our results indicate that charged residues proximal to the seven vestibular portals and their electrostatic interactions with permeating cations determine selectivity and regulate the conductance of MscS and potentially other channels belonging to the MscS subfamily. Furthermore, our findings suggest a role for mechanosensitive channels in bacterial calcium regulation, indicative of functions other than protection against osmolarity changes that these channels possibly fulfil in bacteria.

Insights into the distal heme pocket of H-NOX using fluoride as a probe for H-bonding interactions.

Nitric oxide (NO) and dioxygen (O2) are gases of similar size, shape, and electrostatic potential, but different physiological function. In aerobic organisms, the cellular concentration of O2 far exceeds that of NO; instead NO relies heavily on the ability of its receptor to discriminate against O2. In mammals, soluble guanylate cyclase (sGC) serves this role, binding NO with picomolar sensitivity and excluding O2 binding. Interestingly, some bacterial homologs of sGC, including the H-NOX (heme-nitric oxide/oxygen) domain from Thermoanaerobacter tengcongensis, tightly bind O2. Three distal pocket residues (Trp9, Asn74, and Tyr140) form a hydrogen-bonding network that stabilizes O2 binding to TtH-NOX. Therefore, a current hypothesis to explain sGC ligand specificity is that sGC lacks H-bond donors that preferentially stabilize O2 binding. The wavelength maximum of the charge-transfer band (CT1) in the electronic spectrum of the fluoride complex of ferric hemoproteins is a sensitive probe of H-bonding. Here, in order to gain further understanding of the distal pocket H-bonding network in TtH-NOX, we employ fluoride as a spectroscopic probe. As expected, our results indicate that Y140 donates a strong H-bond to the heme-bound ligand. We find that an H-bond from Asn74 as well as distal pocket crowding contributes to positioning Tyr140 for a strong and directed H-bond to iron-bound ligands; indeed crowding may be the primary role for Trp9. We clarify the role of H-bonding in sGC ligand discrimination and suggest that sterics also regulate ligand binding in the H-NOX family.

Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

[Optimization of titanium dioxide enrichment of phosphopeptides and application in the Thermoanaerobacter tengcongensis phosphoproteome analysis].

Using titanium dioxide is a very good strategy for the phosphopeptide enrichment. There are many other factors can affect the enrichment efficiency, and the optimization of parameters was needed for better enrichment results. In this study, the peptide mixtures of six standard proteins were used as the model samples to evaluate and optimize the parameters such as the proportion of acetonitrile and trifluoroacetic acid in loading buffer and the TiO2-to-peptide ratio. The results showed that 80% (v/v) acetonitrile, 1% (v/v) trifluoroacetic acid and 40 : 1 (m/m) TiO2-to-peptide ratio were the optimum parameters to obtain the best enrichment selectivity and maximum phosphopeptides identification. For the first time, the optimum enrichment conditions were applied for the phosphoproteome analysis of the Thermoanaerobacter tengcongensis, an anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengchong, China, and 25 phosphorylated proteins were identified in the preliminary experiment. The results provided a reference for further study on this organism survived under extreme environment.