Engineering peroxiredoxin 3 to facilitate control over self-assembly.

Oligomeric proteins are abundant in nature and are useful for a range of nanotechnological applications; however, a key requirement in using these proteins is controlling when and how they form oligomeric assemblies. Often, protein oligomerisation is triggered by various cellular signals, allowing for controllable oligomerisation. An example of this is human peroxiredoxin 3 (Prx), a stable protein that natively forms dimers, dodecameric rings, stacks, and tubes in response to a range of environmental stimuli. Although we know the key environmental stimuli for switching between different oligomeric states of Prx, we still have limited molecular knowledge and control over the formation and size of the protein's stacks and tubes. Here, we have generated a range of Prx mutants with either a decreased or knocked out ability to stack, and used both imaging and solution studies to show that Prx stacks through electrostatic interactions that are stabilised by a hydrogen bonding network. Furthermore, we show that altering the length of the polyhistidine tag will alter the length of the Prx stacks, with longer polyhistidine tags giving longer stacks. Finally, we have analysed the effect a variety of heavy metals have on the oligomeric state of Prx, wherein small transition metals like nickel enhances Prx stacking, while larger positively charged metals like tungstate ions can prevent Prx stacking. This work provides further structural characterisation of Prx, to enhance its use as a platform from which to build protein nanostructures for a variety of applications.

Assembly of Protein Stacks With in Situ Synthesized Nanoparticle Cargo.

The ability of proteins to form hierarchical structures through self-assembly provides an opportunity to synthesize and organize nanoparticles. Ordered nanoparticle assemblies are a subject of widespread interest due to the potential to harness their emergent functions. In this work, the toroidal-shaped form of the protein peroxiredoxin, which has a pore size of 7 nm, was used to organize iron oxyhydroxide nanoparticles. Iron in the form of Fe2+ was sequestered into the central cavity of the toroid ring using metal-binding sites engineered there and then hydrolyzed to form iron oxyhydroxide particles bound into the protein pore. By precise manipulation of the pH, the mineralized toroids were organized into stacks confining one-dimensional nanoparticle assemblies. We report the formation and the procedures leading to the formation of such nanostructures and their characterization by chromatography and microscopy. Electrostatic force microscopy clearly revealed the formation of iron-containing nanorods as a result of the self-assembly of the iron-loaded protein. This research bodes well for the use of peroxiredoxin as a template with which to form nanowires and structures for electronic and magnetic applications.

Quaternary structure influences the peroxidase activity of peroxiredoxin 3.

Peroxiredoxins are abundant peroxidase enzymes that are key regulators of the cellular redox environment. A major subgroup of these proteins, the typical 2-Cys peroxiredoxins, can switch between dimers and decameric or dodecameric rings, during the catalytic cycle. The necessity of this change in quaternary structure for function as a peroxidase is not fully understood. In order to explore this, human peroxiredoxin 3 (Prx3) protein was engineered to form both obligate dimers (S75E Prx3) and stabilised dodecameric rings (S78C Prx3), uncoupling structural transformations from the catalytic cycle. The obligate dimer, S75E Prx3, retained catalytic activity towards hydrogen peroxide, albeit significantly lower than the wildtype and S78C proteins, suggesting an evolutionary advantage of having higher order self-assemblies.

Archaeal Lsm rings as stable self-assembling tectons for protein nanofabrication.

We have exploited the self-assembling properties of archaeal-derived protein Lsmα to generate new supramolecular forms based on its stable ring-shaped heptamer. We show that engineered ring tectons incorporating cysteine sidechains on obverse faces of the Lsmα7 toroid are capable of forming paired and stacked formations. A Cys-modified construct, N10C/E61C-Lsmα, appears to organize into disulfide-mediated tube formations up to 45 nm in length. We additionally report fabrication of cage-like protein clusters through conjugation of Cu2+ to His-tagged variants of the Lsmα7 tecton. These 400 kDa protein capsules are seen as cube particles with visible pores, and are reversibly dissembled into their component ring tectons by EDTA. The ß-rich Lsmα supramolecular assemblies described are amenable to further fusion modifications, or for surface attachment, so providing potential for future applications that exploit the RNA-binding capacity of Lsm proteins, such as sensing applications.

Morphology of complexes formed between ß-lactoglobulin nanofibrils and pectins is influenced by the pH and structural characteristics of the pectins.

For the optimal use of ß-lactoglobulin nanofibrils as a raw material in biological composites an in-depth knowledge of their interactions with other constituents is necessary. To understand the effect of electrostatic interactions on the morphology of resulting complexes, ß-lactoglobulin nanofibrils were allowed to interact with pectins in which the amount of available negative charge was controlled by selecting their degree of methylesterification. In this study, citrus pectins having different degrees of methylesterification (∼48, 67, 86, and 97%) were selected and interacted with nanofibrils at pH 2 and pH 3, where they possess a net positive charge. Electrostatic complexes formed between ß-lactoglobulin nanofibrils and all pectin types, except for the sample having a degree of methylesterification of 97%. The morphology of these complexes, however, differed significantly with the degree of methylesterification of the pectin, its concentration, and the pH of the medium, revealing that distinct desired biological architectures can be attained relatively easily through manipulating the electrostatic interactions. Interestingly, the pectin with a degree of methylesterification of 86% was found to crosslink the ß-lactoglobulin nanofibrils into ordered 'nanotapes'.

ß-Lactoglobulin nanofibrils can be assembled into nanotapes via site-specific interactions with pectin.

Controlling the self-assembly of individual supramolecular entities, such as amyloid fibrils, into hierarchical architectures enables the 'bottom-up' fabrication of useful bionanomaterials. Here, we present the hierarchical assembly of ß-lactoglobulin nanofibrils into the form of 'nanotapes' in the presence of a specific pectin with a high degree of methylesterification. The nanotapes produced were highly ordered, and had an average width of 180 nm at pH 3. Increasing the ionic strength or the pH of the medium led to the disassembly of nanotapes, indicating that electrostatic interactions stabilised the nanotape architecture. Small-angle X-ray scattering experiments conducted on the nanotapes showed that adequate space is available between adjacent nanofibrils to accommodate pectin molecules. To locate the interaction sites on the pectin molecule, it was subjected to endopolygalacturonase digestion, and the resulting products were analysed using capillary electrophoresis and size-exclusion chromatography for their charge and molecular weight, respectively. Results suggested that the functional pectin molecules carry short (<10 residues) enzyme-susceptible blocks of negatively charged, non-methylesterified galacturonic acid residues in the middle of their homogalacturonan backbones (and possibly near their ends), that specifically bind to sites on the nanofibrils. Blocking the interaction sites on the nanofibril surface using small oligomers of non-methylesterified galacturonic acid residues similar in size to the interaction sites of the pectin molecule decreased the nanotape formation, indicating that site-specific electrostatic interactions are vital for the cross-linking of nanofibrils. We propose a structural model for the pectin-cross-linked ß-lactoglobulin nanotapes, the elements of which will inform the future design of bionanomaterials.

Proteins as supramolecular building blocks: Nterm-Lsr2 as a new protein tecton.

Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. We have explored self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the N-terminal domain of Lsr2, Nterm-Lsr2. A key feature of this protein is that it undergoes self-assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm-Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm-Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed.

A radish seed antifungal peptide with a high amyloid fibril-forming propensity.

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.

Peroxiredoxin is a versatile self-assembling tecton for protein nanotechnology.

The potential for protein tectons to be used in nanotechnology is increasingly recognized, but the repertoire of stable proteins that assemble into defined shapes in response to an environmental trigger is limited. Peroxiredoxins (Prxs) are a protein family that shows an amazing array of supramolecular assemblies, making them attractive tectons. Human Prx3 (hPrx3) forms toroidal oligomers characteristic of the Prx family, but no structure has been solved to date. Here we report the first 3-D structure of this protein, derived from single-particle analysis of TEM images, establishing a dodecameric structure. This result was supported by SAXS measurements. We also present the first detailed structure of a double toroidal Prx from a higher organism determined by SPA. Guided by these structures, variants of the protein were designed to facilitate controlled assembly of protein nanostructures through the association of the toroids. We observed an enhanced population of stacked toroids, as seen by TEM; nanocages and interlocked toroids were also visible. Low pH was successfully predicted to generate long ordered nanotubes. Control over the length of the tubes was gained by adding ammonium sulfate to the assembly buffer. These versatile assembly properties demonstrate the considerable potential of hPrx3 as a tecton for protein nanotechnology.

A compact microelectrode array chip with multiple measuring sites for electrochemical applications.

In this paper we demonstrate the fabrication and electrochemical characterization of a microchip with 12 identical but individually addressable electrochemical measuring sites, each consisting of a set of interdigitated electrodes acting as a working electrode as well as two circular electrodes functioning as a counter and reference electrode in close proximity. The electrodes are made of gold on a silicon oxide substrate and are passivated by a silicon nitride membrane. A method for avoiding the creation of high edges at the electrodes (known as lift-off ears) is presented. The microchip design is highly symmetric to accommodate easy electronic integration and provides space for microfluidic inlets and outlets for integrated custom-made microfluidic systems on top.

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition.

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.

Dielectrophoretic manipulation and solubility of protein nanofibrils formed from crude crystallins.

Protein nanofibrils and nanotubes are now widely accepted as having potential for use in the field of bionanotechnology. For this to be a feasible alternative to existing technologies, there is a need for a commercially viable source. Previous work has identified amyloid fibrils formed from crude crystallin proteins as such a source, since these fibrils can be produced in large quantities at a low cost. Applications include use of fibrils as templates for the formation of nanowires or as biosensing scaffolds. There remains a number of practical considerations, such as stability and the ability to control their arrangement. In this study, crude crystallin amyloid fibrils are shown to be stable in a range of biological and clean room solvents, with the fibril presence confirmed by transmission electron microscopy and the thioflavin T fluorescent assay. The fibrils were also immobilised between microelectrodes using dielectrophoresis, which enabled the recording of I-V curves for small numbers of fibrils. This investigation showed the fibrils to have low conductivity, with current values in the range of 10(-10) A recorded. This low conductivity could be increased through modification, or alternately, the fibrils could be used unmodified for applications where they can act as templates or high surface area nanoscaffolds.

Aggregation and fibrillogenesis of proteins not associated with disease: a few case studies.

While amyloid structures have been well characterised in a medical context, there is increasing interest in studying amyloid-like aggregates in other areas, such as food science and nanomaterials. Several proteins relevant to food processing, including serum albumen, lactoglobulin, lysozyme, ovalbumin, casein, and soy protein isolate have been shown to form fibrillar structures under both physiological and non-physiological conditions. These structures are likely to contribute to the structural characteristics of the final food product. In a biotechnological context, proteins such as insulin and eye lens crystallins can be induced to form amyloid structures which can subsequently be used in biotechnology. One example of this is the use of amyloid fibrils as a scaffold for the immobilisation of enzymes. Another current interest in amyloid fibrils is as a storage form for peptide hormones, including insulin, glucagon and calcitonin. Here, we give an overview of a selection of well characterised proteins that have been studied outside the context of disease.

Insight into the self-assembly and gel formation of a bioactive peptide derived from bovine casein.

Abstract: The self-assembling and gelation properties of a bioactive peptide derived from bovine casein (FFVAPFPEVFGK) were studied in the peptide's natural form (uncapped, uncapFFV) and capped with protecting groups added to both termini (capped, capFFV). Although the natural peptide (uncapFFV) did not demonstrate self-assembly, the capped peptide (capFFV) spontaneously self-assembled and formed a self-supporting gel. Variations in peptide concentration and incubation time influenced the gel's mechanical properties, suggesting the peptide's properties could be tuned and exploited for different applications. These results suggest that food-derived bioactive peptides have good potential for self-assembly and therefore utilisation as gels in functional foods and nutraceuticals. Background: Self-assembly is a natural phenomenon that occurs in many fundamental biological processes. Some peptides can self-assemble and form gels with tunable properties under given conditions. These properties, along with peptide bioactivity, can be combined to make unique biomaterials. Instead of synthesising the self-assembling bioactive peptides, we aim to extract them from natural sources. In order to use these peptides for different applications, it is essential to understand how we can trigger self-assembly and optimise the assembly conditions of these peptide gels. Scope: The self-assembling and gelation properties of a bioactive peptide derived from bovine casein (FFVAPFPEVFGK) were studied in the peptide's natural form (uncapped, uncapFFV) and capped with protecting groups added to both termini (capped, capFFV). Major conclusions: Although the natural peptide (uncapFFV) did not demonstrate self-assembly, the capped peptide (capFFV) spontaneously self-assembled and formed a self-supporting gel. Variations in peptide concentration and incubation time influenced the gel's mechanical properties, suggesting the peptide's properties could be tuned and exploited for different applications. General significance: These results suggest that food-derived bioactive peptides have good potential for self-assembly and therefore utilisation as gels in functional foods and nutraceuticals.

Characterizing lysinoalanine crosslinks in food systems: Discovery of a diagnostic ion in model peptides using MALDI mass spectrometry.

Formation of lysinoalanine protein-protein crosslinks during food processing adversely impacts nutritional value. However, mapping lysinoalanine directly in food is challenging. We characterized the fragmentation pattern of lysinoalanine crosslinks in synthetic peptide models over a range of pH and time treatments using mass spectrometry. A putative diagnostic ion resulting from the cleavage of the α-carbon and ß-carbon of lysinoalanine is identified in MALDI MS/MS spectra. This represents the first step in mapping lysinoalanine in real food samples with higher precision than currently identifiable through standard or customized software. We then determined a correlated trend in the reduction of disulfide bonds and formation of lysinoalanine with increasing pH and time. Mapping lysinoalanine formation is critical to enhance our understanding of molecular processes impacting the nutritional value of foods, including notably in the development of protein alternatives that use alkaline treatment to extract protein isolates.

The Leptospermum scoparium (Manuka)-Specific Nectar and Honey Compound 3,6,7-Trimethyllumazine (LepteridineTM) That Inhibits Matrix Metalloproteinase 9 (MMP-9) Activity.

3,6,7-trimethyllumazine (Lepteridine™) is a newly discovered natural pteridine derivative unique to Manuka (Leptospermum scoparium) nectar and honey, with no previously reported biological activity. Pteridine derivative-based medicines, such as methotrexate, are used to treat auto-immune and inflammatory diseases, and Manuka honey reportedly possesses anti-inflammatory properties and is used topically as a wound dressing. MMP-9 is a potential candidate protein target as it is upregulated in recalcitrant wounds and intestinal inflammation. Using gelatin zymography, 40 µg/mL LepteridineTM inhibited the gelatinase activities of both pro- (22%, p < 0.0001) and activated (59%, p < 0.01) MMP-9 forms. By comparison, LepteridineTM exerted modest (~10%) inhibition against a chromogenic peptide substrate and no effect against a fluorogenic peptide substrate. These findings suggest that LepteridineTM may not interact within the catalytic domain of MMP-9 and exerts a negligible effect on the active site hydrolysis of small soluble peptide substrates. Instead, the findings implicate fibronectin II domain interactions by LepteridineTM which impair gelatinase activity, possibly through perturbed tethering of MMP-9 to the gelatin matrix. Molecular modelling analyses were equivocal over interactions at the S1' pocket versus the fibronectin II domain, while molecular dynamic calculations indicated rapid exchange kinetics. No significant degradation of synthetic or natural LepteridineTM in Manuka honey occurred during simulated gastrointestinal digestion. MMP-9 regulates skin and gastrointestinal inflammatory responses and extracellular matrix remodelling. These results potentially implicate LepteridineTM bioactivity in Manuka honey's reported beneficial effects on wound healing via topical application and anti-inflammatory actions in gastrointestinal disorder models via oral consumption.

Characterization of monomeric dihydrodipicolinate synthase variant reveals the importance of substrate binding in optimizing oligomerization.

To gain insights into the role of quaternary structure in the TIM-barrel family of enzymes, we introduced mutations to the DHDPS enzyme of Thermotoga maritima, which we have previously shown to be a stable tetramer in solution. These mutations were aimed at reducing the number of salt bridges at one of the two tetramerization interface of the enzyme, which contains many more interactions than the well characterized equivalent interface of the mesophilic Escherichia coli DHDPS enzyme. The resulting variants had altered quaternary structure, as shown by analytical ultracentrifugation, gel filtration liquid chromatography, and small angle X-ray scattering, and X-ray crystallographic studies confirmed that one variant existed as an independent monomer, but with few changes to the secondary and tertiary structure. Reduction of higher order assembly resulted in a loss of thermal stability, as measured by a variety of methods, and impaired catalytic function. Binding of pyruvate increased the oligomeric status of the variants, with a concomitant increase in thermal stability, suggesting a role for substrate binding in optimizing stable, higher order structures. The results of this work show that the salt bridges located at the tetramerization interface of DHDPS play a significant role in maintaining higher order structures, and demonstrate the importance of quaternary structure in determining protein stability and in the optimization of enzyme catalysis.

Formation of ß-lactoglobulin nanofibrils by microwave heating gives a peptide composition different from conventional heating.

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of ß-lactoglobulin (ß-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of ß-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact ß-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of ß-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of ß-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.

Aspects of physical and chemical alterations to proteins during food processing - some implications for nutrition.

In this paper, we give an overview of our research exploring the impact of physical and chemical processing on food proteins. There are three themes, applied to the proteins of wheat, soya, egg and dairy foods. Firstly, the impact of the Maillard reaction on food proteins is discussed, with a particular focus on how the reactions might be harnessed to manipulate food texture. Secondly, the potential of enzymatic protein-protein crosslinking is considered, especially the enzyme transglutaminase. Thirdly, the broader question of how the aggregation of proteins within a food is altered by chemical and physical modification and how, in turn, this might impact on the overall nutritional quality of the food is considered.

1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase.

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were synthesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight binding inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model of inhibition, demonstrating either an encounter complex or a condensation product for the slow and slow-tight binding inhibitors, respectively.