Solution characterization of the dynamic conjugative entry exclusion protein TraG.

The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor-donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding-mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.

Biomedical Applications of Biomolecules Isolated from Methanotrophic Bacteria in Wastewater Treatment Systems.

Wastewater treatment plants and other remediation facilities serve important roles, both in public health, but also as dynamic research platforms for acquiring useful resources and biomolecules for various applications. An example of this is methanotrophic bacteria within anaerobic digestion processes in wastewater treatment plants. These bacteria are an important microbial source of many products including ectoine, polyhydroxyalkanoates, and methanobactins, which are invaluable to the fields of biotechnology and biomedicine. Here we provide an overview of the methanotrophs' unique metabolism and the biochemical pathways involved in biomolecule formation. We also discuss the potential biomedical applications of these biomolecules through creation of beneficial biocompatible products including vaccines, prosthetics, electronic devices, drug carriers, and heart stents. We highlight the links between molecular biology, public health, and environmental science in the advancement of biomedical research and industrial applications using methanotrophic bacteria in wastewater treatment systems.

Screening for Methane Utilizing Mixed Communities with High Polyhydroxybutyrate (PHB) Production Capacity Using Different Design Approaches.

With the adverse environmental ramifications of the use of petroleum-based plastic outweighing the challenges facing the industrialization of bioplastics, polyhydroxyalkanoate (PHA) biopolymer has gained broad interest in recent years. Thus, an efficient approach for maximizing polyhydroxybutyrate (PHB) polymer production in methanotrophic bacteria has been developed using the methane gas produced in the anaerobic digestion process in wastewater treatment plants (WWTPS) as a carbon substrate and an electron donor. A comparison study was conducted between two experimental setups using two different recycling strategies, namely new and conventional setups. The former setup aims to recycle PHB producers into the system after the PHB accumulation phase, while the latter recycles the biomass back into the system after the exponential phase of growth or the growth phase. The goal of this study was to compare both setups in terms of PHB production and other operational parameters such as growth rate, methane uptake rate, and biomass yield using two different nitrogen sources, namely nitrate and ammonia. The newly proposed setup is aimed at stimulating PHB accumulating type II methanotroph growth whilst enabling other PHB accumulators to grow simultaneously. The success of the proposed method was confirmed as it achieved highest recorded PHB accumulation percentages for a mixed culture community in both ammonia- and nitrate-enriched media of 59.4% and 54.3%, respectively, compared to 37.8% and 9.1% for the conventional setup. Finally, the sequencing of microbial samples showed a significant increase in the abundance of type II methanotrophs along with other PHB producers, confirming the success of the newly proposed technique in screening for PHB producers and achieving higher PHB accumulation.

Protein Dynamics in F-like Bacterial Conjugation.

Efficient in silico development of novel antibiotics requires high-resolution, dynamic models of drug targets. As conjugation is considered the prominent contributor to the spread of antibiotic resistance genes, targeted drug design to disrupt vital components of conjugative systems has been proposed to lessen the proliferation of bacterial antibiotic resistance. Advancements in structural imaging techniques of large macromolecular complexes has accelerated the discovery of novel protein-protein interactions in bacterial type IV secretion systems (T4SS). The known structural information regarding the F-like T4SS components and complexes has been summarized in the following review, revealing a complex network of protein-protein interactions involving domains with varying degrees of disorder. Structural predictions were performed to provide insight on the dynamicity of proteins within the F plasmid conjugative system that lack structural information.

Crystal structures of the synthetic inter-mediate 3-[(6-chloro-7H-purin-7-yl)meth-yl]cyclo-butan-1-one, and of two oxetanocin derivatives: 3-[(6-chloro-8,9-di-hydro-7H-purin-7-yl)meth-yl]cyclo-butan-1-ol and 3-[(6-chloro-9H-purin-9-yl)meth-yl]cyclo-butan-1-ol.

The crystal structures of an inter-mediate, C10H9ClN4O, 3-[(6-chloro-7H-purin-7-yl)meth-yl]cyclo-butan-1-one (I), and two N-7 and N-9 regioisomeric oxetanocin nucleoside analogs, C10H13ClN4O, 3-[(6-chloro-8,9-di-hydro-7H-purin-7-yl)meth-yl]cyclo-butan-1-ol (II) and C10H11ClN4O, 3-[(6-chloro-9H-purin-9-yl)meth-yl]cyclo-butan-1-ol (IV), are reported. The crystal structures of the nucleoside analogs confirmed the reduction of the N-7- and N-9-substituted cyclo-butano-nes with LiAl(OtBu)3 to occur with facial selectivity, yielding cis-nucleosides analogs similar to those found in nature. Reduction of the purine ring of the N-7 cyclo-butanone to a di-hydro-purine was observed for compound (II) but not for the purine ring of the N-9 cyclo-butanone on formation of compound (IV). In the crystal of (I), mol-ecules are linked by a weak Cl⋯O inter-action, forming a 21 helix along [010]. The helices are linked by offset π-π inter-actions [inter-centroid distance = 3.498 (1) Å], forming layers parallel to (101). In the crystal of (II), mol-ecules are linked by pairs of O-H⋯N hydrogen bonds, forming inversion dimers with an R 2 2(8) ring motif. The dimers are linked by O-H⋯N hydrogen bonds, forming chains along [001], which in turn are linked by C-H⋯π and offset π-π inter-actions [inter-centroid distance = 3.509 (1) Å], forming slabs parallel to the ac plane. In the crystal of (IV), mol-ecules are linked by O-H⋯N hydrogen bonds, forming chains along [101]. The chains are linked by C-H⋯N and C-H⋯O hydrogen bonds and C-H⋯π and offset π-π inter-actions [inter-centroid distance = 3.364 (1) Å], forming a supra-molecular framework.

Structural and functional consequences of the STAT5BN642H driver mutation.

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

Protein Nanotubes: From Bionanotech towards Medical Applications.

Nanobiotechnology involves the study of structures found in nature to construct nanodevices for biological and medical applications with the ultimate goal of commercialization. Within a cell most biochemical processes are driven by proteins and associated macromolecular complexes. Evolution has optimized these protein-based nanosystems within living organisms over millions of years. Among these are flagellin and pilin-based systems from bacteria, viral-based capsids, and eukaryotic microtubules and amyloids. While carbon nanotubes (CNTs), and protein/peptide-CNT composites, remain one of the most researched nanosystems due to their electrical and mechanical properties, there are many concerns regarding CNT toxicity and biodegradability. Therefore, proteins have emerged as useful biotemplates for nanomaterials due to their assembly under physiologically relevant conditions and ease of manipulation via protein engineering. This review aims to highlight some of the current research employing protein nanotubes (PNTs) for the development of molecular imaging biosensors, conducting wires for microelectronics, fuel cells, and drug delivery systems. The translational potential of PNTs is highlighted.

Structural flexibility in the Helicobacter pylori peptidyl-prolyl cis,trans-isomerase HP0175 is achieved through an extension of the chaperone helices.

Helicobacter pylori infects the gastric epithelium of half the global population, where infections can persist into adenocarcinomas and peptic ulcers. H. pylori secretes several proteins that lend to its pathogenesis and survival including VacA, CagA, γ-glutamyltransferase and HP0175. HP0175, also known as HpCBF2, classified as a peptidyl-prolyl cis,trans-isomerase, has been shown to induce apoptosis through a cascade of mechanisms initiated though its interaction with toll like receptor 4 (TLR4). Here, we report the structure of apo-HP0175 at 2.09 Šwith a single monomer in the asymmetric unit. Chromatographic, light scattering and mass spectrometric analysis of HP0175 in solution indicate that the protein is mainly monomeric under low salt conditions, while increasing ionic interactions facilitates protein dimerization. A comparison of the apo-HP0175 structure to that of the indole-2-carboxylic acid-bound form shows movement of the N- and C-terminal helices upon interaction of the catalytic residues in the binding pocket. Helix extension of the N/C chaperone domains between apo and I2CA-bound HP0175 supports previous findings in parvulin PPIases for their role in protein stabilization (and accommodation of variable protein lengths) of those undergoing catalysis.

The interaction of TraW and TrbC is required to facilitate conjugation in F-like plasmids.

Bacterial conjugation, such as that mediated by the E. coli F plasmid, is a main mechanism driving bacterial evolution. Two important proteins required for F-pilus assembly and DNA transfer proficiency are TraW and TrbC. As members of a larger complex, these proteins assemble into a type IV secretion system and are essential components of pore formation and mating pair stabilization between the donor and the recipient cells. In the current report, we demonstrate the physical interaction of TraW and TrbC, show that TraW preferentially interacts with the N-terminal domain of TrbC, and that this interaction is important in restoring conjugation in traW/trbC knockouts.

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

HDX-MS and deletion analysis of the type 4 secretion system protein TraF from the Escherichia coli F plasmid.

Conjugative DNA transfer by the F-plasmid is achieved through a type IV secretion system (T4SS) encoded within the plasmid's transfer region; TraF is one of several F-T4SS proteins essential for F-pilus assembly. In order to identify regions of the protein important for TraF function, a series of deletion mutants were assessed for their ability to recover conjugative transfer in a traF knockout. Interestingly, modification of any region of TraF abolishes pilus synthesis, resulting in a loss of rescue of conjugative function. Dynamic analysis of TraF by time-resolved hydrogen-deuterium exchange revealed that the C-terminal region containing the predicted thioredoxin-like domain is quite structured, while the N-terminal region, predicted to interact with TraH in the intact F-T4SS, was more dynamic.

Dimerization of the type IV pilin from Pseudomonas aeruginosa strain K122-4 results in increased helix stability as measured by time-resolved hydrogen-deuterium exchange.

Truncated pilin monomers from Pseudomonas aeruginosa strain K122-4 (ΔK122) have been shown to enter a monomer-dimer equilibrium in solution prior to oligomerization into protein nanotubes. Here, we examine the structural changes occurring between the monomeric and dimeric states of ΔK122 using time-resolved hydrogen-deuterium exchange mass spectrometry. Based on levels of deuterium uptake, the N-terminal α-helix and the loop connecting the second and third strands of the anti-parallel ß-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel ß-sheet and αß loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state.

Expression, purification, crystallization and preliminary X-ray analysis of the receiver domain of Staphylococcus aureus LytR protein.

The response-regulatory protein LytR belongs to a family of transcription factors involved in the regulation of important virulence factors in pathogenic bacteria. The protein consists of a receiver domain and an effector domain, which play an important role in controlled cell death and lysis. The LytR receiver domain (LytR(N)) has been overexpressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The crystals grew as needles, with unit-cell parameters a = b = 84.82, c = 157.3 Å, α = ß = 90, γ = 120°. LytR(N) crystallized in space group P6122 and the crystals diffracted to a maximum resolution of 2.34 Å. Based on the Matthews coefficient (V(M) = 5.44 Å(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit.

Fibril-mediated oligomerization of pilin-derived protein nanotubes.

BACKGROUND: Self-assembling protein nanotubes (PNTs) are an intriguing alternative to carbon nanotubes for applications in bionanotechnology, in part due to greater inherent biocompatibility. The type IV pilus of the gram negative bacteria Pseudomonas aeruginosa is a protein-based fibre composed of a single subunit, the type IV pilin. Engineered pilin monomers from P. aeruginosa strain K122-4 (ΔK122) have been shown to oligomerize into PNTs both in solution and at surfaces. In order to fully exploit PNTs in bionanotechonological settings, an in-depth understanding of their assembly, physical characteristics and robustness, both in solution and when constrained to surfaces, is required. RESULTS: This study details the effectiveness of multiple initiators of ΔK122-derived PNT oligomerization and characterize the formation of PNTs in solution. The optimal initiator for the oligomerization of ΔK122 in solution was observed to be 2-methyl-2,4-pentanediol (MPD). Conversely, larger PEG molecules do not trigger oligomerization. Multi-angle light scattering analysis indicates that the pilin protein exists in a monomer-dimer equilibrium in solution, and that an intermediate species forms within three hours that then coalesces over time into high molecular weight PNTs. Transmission Electron Microscopic analysis was used to observe the formation of oligomerized ΔK122 fibrils prior to assembly into full-length PNTs. CONCLUSIONS: The oligomerization of ΔK122 pilin derived PNTs is a fibril mediated process. The optimal trigger for PNT oligomerization in solution is MPD, and the observation that PEGs do not induce oligomerization may enable the oligomerization of pilin-derived PNTs on PEG-functionalized surfaces for implantable bionanodevices.

Cross-linked glucose oxidase clusters for biofuel cell anode catalysts.

The efficient localization of increased levels of active enzymes onto conducting scaffolds is important for the development of enzyme-based biofuel cells. Cross-linked enzyme clusters (CEC) of glucose oxidase (GOx) constrained to functionalized carbon nanotubes (CEC-CNTs) were generated in order to evaluate the potential of using CECs for developing GOx-based bioanodes functioning via direct electron transfer from the GOx active site to the CNT scaffold. CEC-CNTs generated from several weight-to-weight ratios of GOx:CNT were examined for comparable catalytic activity to free GOx into the solution, with CEC-CNTs generated from a 100% GOx solution displaying the greatest enzymatic activity. Scanning transmission electron microscopic analysis of CEC-CNTs generated from 100% GOx to CNT (wt/wt) ratios revealed that CEC clusters of ∼78 µm2 localized to the CNT surface. Electrochemical analysis indicates that the enzyme is engaged in direct electron transfer, and biofuel cells generated using GOx CEC-CNT bioanodes were observed to have a peak power density of ∼180 µW cm(-2). These data indicate that the generation of nano-to-micro-sized active enzyme clusters is an attractive option for the design of enzyme-specific biofuel cell powered implantable devices.

Peptide and protein-based nanotubes for nanobiotechnology.

The development of biologically relevant nanosystems such as biomolecular probes and sensors requires systems that effectively interface specific biochemical environments with abiotic architectures. The most widely studied nanomaterial, carbon nanotubes, has proven challenging in their adaptation for biomedical applications despite their numerous advantageous physical and electrochemical properties. On the other hand, development of bionanosystems through adaptation of existing biological systems has several advantages including their adaptability through modern recombinant DNA strategies. Indeed, the use of peptides, proteins and protein assemblies as nanotubes, scaffolds, and nanowires has shown much promise as a bottom-up approach to the development of novel bionanosystems. We highlight several unique peptide and protein systems that generate protein nanotubes (PNTs) that are being explored for the development of biosensors, probes, bionanowires, and drug delivery systems.

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism.

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.

Development of protein nanotubes from a multi-purpose biological structure.

One approach to develop nanosystems that incorporate biological concepts involves the addition of biotic moieties (carbohydrates, DNA, protein) to abiotic scaffolds such as carbon nanotubes. These hybrids have interesting properties but incorporation of specific, site-directed functionalization is challenging and the resulting material is best described in terms of its bulk properties. An alternative approach to the development of bionanosystems is to adapt an existing biological system. This method has several advantages, including access to the powerful tools of protein engineering and ready biological acceptance as these structures themselves are biotic in origin. We have chosen the type IV pilus, a fiber-like structure from the bacteria Pseudomonas aeruginosa, as our model system for the development of a protein-based nanotube. This review highlights the biological characteristics of our model system, presents the novel features of our pilin-derived protein nanotubes, and discusses how these protein nanotubes may contribute to bionanotechnology.

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient.

The mating pair stabilization (Mps) protein of the F plasmid, TraG, is unique to F-like type IV secretion systems. TraG is a polytopic inner-membrane protein with a large C-terminal periplasmic domain that is required for piliation and Mps, whereas the N-terminal region is sufficient for pilus synthesis. The C-terminal region of TraG is thought to be cleaved by the host signal peptidase I to give a fragment called TraG* that is responsible for Mps. Using mutational analysis and cell localization studies, it was shown that TraG* is most probably an artifact caused by non-specific degradation. TraS (173 aa in F), which is involved in entry exclusion (Eex), blocks redundant conjugative DNA synthesis and transport between donor cells, suggesting that it interferes with a signalling pathway required to trigger DNA transfer. Using the F and R100 plasmids, TraG in the donor cell was found to recognize TraS in the recipient cell inner membrane, in a plasmid-specific manner. This activity mapped to aa 610-673 in F TraG, the only region that differs significantly from R100 TraG. Expression of traG or traG* in a recipient cell did not affect mating ability or Eex. These results suggest that TraG may be translocated to the recipient cell, where it contacts the inner membrane, initiating transfer, a process that is blocked by TraS.

Purification, kinetic characterization, and mapping of the minimal catalytic domain and the key polar groups of Helicobacter pylori alpha-(1,3/1,4)-fucosyltransferases.

The minimal catalytic domain of alpha-(1,3/1,4)-fucosyltransferases (FucTs) from Helicobacter pylori strains NCTC11639 and UA948 was mapped by N- and C-terminal truncations. Only the C terminus could be truncated without significant loss of activity. 11639FucT and UA948FucT contain 10 and 8 heptad repeats, respectively, which connect the catalytic domain with the C-terminal putative amphipathic alpha-helices. Deletion of all heptad repeats almost completely abolished enzyme activity. Nevertheless, with only one heptad repeat 11639FucT is fully active, whereas UA948FucT is partially active. Removal of the two putative amphipathic alpha-helices dramatically increased protein expression and solubility, enabling purification with yields of milligrams/liter. Steady-state kinetic analysis of the purified FucTs showed that 11639FucTs possessed slightly tighter binding affinity for both Type II acceptor and GDP-fucose donor than UA948FucT, and its kcat of 2.3 s(-1) was double that of UA948FucT, which had a kcat value of 1.1 s(-1) for both Type II and Type I acceptors. UA948FucT strongly favors Type II over the Type I acceptor with a 20-fold difference in acceptor Km. Sixteen modified Type I and Type II series acceptors were employed to map the molecular determinants of acceptors required for recognition by H. pylori alpha-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in alpha1,3 activity, whereas in Type I acceptor this completely abolished alpha1,4 activity, indicating that this hydroxyl group is a key polar group.