Gerald F. Audette

The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces

Pseudomonas aeruginosa readily binds to stainless steel and other abiotic surfaces, causing major problems in both the medical and food industries. In this study, we show that P. aeruginosa binds to abiotic surfaces in a concentration‐dependent, saturable manner during the initial stages of biofilm formation. P. aeruginosa type IV pili mediate binding to stainless steel as a pilus‐deficient strain does not bind to steel, purified type IV pili bound in a concentration‐dependent, saturable manner, and purified pili competitively inhibited whole cell binding. PAK pili can also bind polystyrene and polyvinylchloride in a concentration‐dependant and saturable manner. As an antibody specific for the C‐terminal pilin receptor binding domain inhibited adherence to abiotic surfaces, the role of the C‐terminal receptor binding domain in mediating binding to steel surfaces was examined. A synthetic peptide of the PAK pilin epithelial cell receptor binding domain [PAK(128–144)ox] bound directly to steel with high affinity. The interaction of pili with steel was specifically inhibited by this peptide with an apparent Ki of ∼0.2 nM and effectively inhibited the binding of viable homologous and heterologous P. aeruginosa strains to steel with an apparent Ki of ∼4 nM. A single point mutation (K130I) in the PAO receptor binding domain was observed to abolish binding to stainless steel while binding to human buccal epithelial cells was enhanced. Therefore, the C‐terminal receptor binding domain appears to have evolved for binding a variety of surfaces.

DNA Binding: a Novel Function of Pseudomonas aeruginosa Type IV Pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.

Hyperphosphorylation of intrinsically disordered tau protein induces an amyloidogenic shift in its conformational ensemble.

Tau is an intrinsically disordered protein (IDP) whose primary physiological role is to stabilize microtubules in neuronal axons at all stages of development. In Alzheimers and other tauopathies, tau forms intracellular insoluble amyloid aggregates known as neurofibrillary tangles, a process that appears in many cases to be preceded by hyperphosphorylation of tau monomers. Understanding the shift in conformational bias induced by hyperphosphorylation is key to elucidating the structural factors that drive tau pathology, however, as an IDP, tau is not amenable to conventional structural characterization. In this work, we employ a straightforward technique based on Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) and Hydrogen/Deuterium Exchange (HDX) to provide a detailed picture of residual structure in tau, and the shifts in conformational bias induced by hyperphosphorylation. By comparing the native and hyperphosphorylated ensembles, we are able to define specific conformational biases that can easily be rationalized as enhancing amyloidogenic propensity. Representative structures for the native and hyperphosphorylated tau ensembles were generated by refinement of a broad sample of conformations generated by low-computational complexity modeling, based on agreement with the TRESI-HDX profiles.

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.

Purification, Kinetic Characterization, and Mapping of the Minimal Catalytic Domain and the Key Polar Groups of Helicobacter pylori α-(1,3/1,4)-Fucosyltransferases

The minimal catalytic domain of α-(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 α-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 α-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 α-(1,3/1,4)-FucTs. Deoxygenation at 6-C of the galactose in Type II acceptor caused a 5000-fold decrease in α1,3 activity, whereas in Type I acceptor this completely abolished α1,4 activity, indicating that this hydroxyl group is a key polar group.

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.

Protein hot spots at bio-nano interfaces

Nanotechnology has influenced the direction of research across the sciences, medicine, and engineering. Carbon nanotubes (CNTs) and, more recently, protein nanotubes (PNTs) and protein-inorganic nanocomposites have received considerable attention due to their unique nanostructures that can be utilized as a scaffold to house proteins or create nanowires. A shift towards protein-inorganic interactions has numerous applications from biosensors to biofuel cells and bio-based nanodevices. We examine several systems where protein hot spots, the active domains on proteins and the interactive dynamics in them, play a critical role in the interactions at the interface of these unique systems.

Mapping Protein: Carbohydrate Interactions

Many biologically important interactions occur between proteins and carbohydrates. The examination of these interactions at the atomic level is critical not only in understanding the nature of these interactions and their biological role, but also in the design of effective modulators of these interactions. While experimentally obtained structural information is preferred, quite often this information is unavailable. In order to address this, several methods have been developed to probe the interactions between protein and carbohydrate in the absence of structural data. These methods map the interactions between protein and carbohydrate, and identify the groups involved, both at the carbohydrate and protein level. Here, we review these developments, and examine the strengths, weaknesses, and pitfalls of these methods.

Fibril-mediated oligomerization of pilin-derived protein nanotubes

BackgroundSelf-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.ResultsThis 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.ConclusionsThe 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.