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. © 2012 Wiley Periodicals, Inc. How to cite this article: WIREs Nanomed Nanobiotechnol 2012, 4:575-585. doi: 10.1002/wnan.1180 INTRODUCTION T he rapidly evolving field of nanotechnology presents constant demands on the scientific community to advance and create new materials to be used in medicine (diagnostics, drug delivery, and tissue engineering), electronics (memory storage, nanoelectronics, quantum computers, novel semiconductor, and optoelectronic devices), bioseparation, catalysis etc. The most studied nanomaterial is carbon nanotubes (CNTs), which possess highly desirable properties including excellent tensile strength, large surface area, favorable electronic, thermal and chemical characteristics. 1 It is these properties that make CNTs interesting vehicles for a variety of areas such as delivery of genes and drugs into bodies, biosensors, and as scaffolds for engineered tissues. However, there are numerous concerns about the toxicity and biodegradability of CNTs, 1-4 adverse effects to electrical properties 5 as a result of exposure to humidity, oxygen, N 2 O, and NH 3 , as well the lack of solubility in aqueous solutions present significant challenges for the use of CNTs in biological and/or biomedical applications. * Correspondence to: [email protected] Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada One approach to mitigating the challenges of CNT solubility and cytotoxicity is to modify the CNTs with biomolecules. For instance, some groups have modified CNTs with chitosan derivatives or incorporated them into a chitosan matrix 6,7 or coated CNTs with proteins 8−10 to improve biocompatibility for tissue engineering. 3,4 While these approaches show distinct promise, a separate approach is to make the nanotubes entirely of peptides or proteins. Protein-based nanotubes can be particularly desirable for biomedical applications due to, for example, assembly under physiologically-relevant conditions as well as their ease of functionalization through protein engineering approaches. We focus on several interesting peptide and protein nanotube (PNT) systems that are showing promise as nanotubes for biomedical applications. PEPTIDE NANOTUBES Peptides, being much shorter than their protein counterparts, often lack the secondary and tertiary structures (i.e., α-helices, β-sheets) associated with proteins. This general lack of initial secondary structure makes peptides an interesting choice for nanotube development, primarily through the induction of secondary structure upon nanotube formation. Indeed, 13 Other groups have also demonstrated the assembly of nanostructures from a variety of peptide modalities including fluorescently labeled dipeptides, 14 tripeptides, The assembly of peptide nanotubes under facile conditions suggests that they may be adapted as scaffolds for biologically based nanowires. Indeed, Reches and Gazit 20 demonstrated the potential for creation of a peptide-based nanowire. In this study, the authors found that a Phe-Phe dipeptide from an amyloidogenic hexapeptide provided the necessary attractive forces to generate stable micrometerlength nanotubes. The peptide nanotubes provided an internal pore that could be filled with an ionic silver solution; the peptide could then be removed through digestion with proteinase K, resulting in a bare silver nanowire. 20 These data demonstrate that the peptide nanotube could be used as a casting mould for metal nanowires, as well as a biological encasement for the same nanowires, depending on whether the peptide is digested away or not. Further studies into these peptide nanotubes has shown that these peptide precursors can assemble into different nanostructures depending on the treatment of the peptide prior to initiation of oligomerization, 21,22 and that they exhibit significant piezoelectricity 23 and photoluminescence. 24,25 The development of biomedical devices incorporating these peptide nanotube based devices shows distinct promise, either on their own or as casting moulds for metal nanowires. PROTEIN NANOTUBES The increased complexity inherent in fully folded proteins, that is their native tertiary structure, presents an interesting challenge in the development of these systems as nanotubes. The question of how does one use a protein's native structure to assemble into a nanotube, either via a patterned template or through self-assembly is of central importance in developing a PNT. Because of the inherent challenges associated with de novo design of PNTs from fully folded proteins, several groups have undertaken an adaptation bacterial flagella and pili, the fiber-like protein polymers produced by many bacteria for a variety of functions. Outlined below are several examples of flagella and pilin-based PNTs, as well as several other protein-based PNTs. Flagella-Based PNTs Flagella, fiber-like structures produced by bacteria for cellular motility, are structurally composed of three general multi-protein components: a proton gradient-driven motor complex, a joint structure, and long helical fiber. Native flagella are 10-15 μm in length with inner and outer diameters of 2-3 nm and 12-25 nm, respectively; 26 the helical fiber is generated from thousands of copies of the FliC (flagelin) protein. In 2006, Kumara et al. reported the self-assembly of a flagellin-based PNT using a FliC-thioredoxin fusion protein, denoted FliTrx. 27 The FliTrx construct fused 109 thioredoxin residues between Gly-243 and Ala-352 of FliC such that the thioredoxin active site was readily accessible by several loop peptides designed to be presented on the PNT surface. FliTrx PNTs were observed through fluorescence microscopy to form 4-10 μm bundles ( 29 The flagellin subunit FliC has also been utilized as a potential vector for liposome-based drug delivery. In particular Ngweniform et al. assembled FliC-based PNTs, which display an overall anionic surface, for coordination of cationic liposomes. 30 The 30 Pilin-Based PNTs Type IV Pili (T4P) are flexible hair-like structures produced at the poles of many gram-negative bacteria. Native T4P are 6 nm in diameter and have a length of up to several micrometers, which the bacteria can control by retraction and extension of pili via a complex type II secretion system, 56 Preassembled pilin-based PNTs have been observed to bind to stainless steel in a manner similar to the binding observed for T4P, In addition to PNTs generated from engineered pilin monomers, functional nanostructures have been generated from native bacterial pili. While not technically nanotubes as the native pilus is itself a nanofiber, these structures are indeed interesting and show distinct promise for application development. For instance, the type IV pili of Geobacter sulfurreducens were shown to mediate the reduction of Fe(III) oxides, OTHER PROTEIN NANOTUBES Self-Assembled PNTs There are several examples of self-assembled PNTs generated from non-pilin or flagellar proteins. For instance, Ballister and colleagues reported PNT selfassembly from Hcp1, a 17.4 kDa protein of the 578 © 2012 Wiley Periodicals, Inc. Volume 4, September/October 2012 WIREs Nanomedicine and Nanobiotechnology Peptide and protein-based nanotubes for nanobiotechnology P. aeruginosa type IV secretion system. 66 Hcp1-derived PNTs adopt a hexameric ring structure with lengths of up to 100 nm (∼25 4.4 nm high subunit rings), an outer diameter of 9.0 nm, a height of 4.4 nm, and an inner cavity of 4.0 nm. The Hcp1 protein rings in the PNT stack non-helically and are held together by mechanical compatibility and engineered disulfidebonding through G90S and R157S mutations. An intriguing aspect of this study in the ability to control PNT length through introduction of chain-terminating subunits, as well as the capability to distinguish PNT polarity by incorporation of uniquely engineered protein caps. 67 TRAP monomers form 11-mer rings that polymerize into PNTs of approximately 8 nm in diameter. 67 A third example of a non-pilin PNT is the observed self-assembly of the milk protein α-lactalbumin, 68 While this result may affect PNT homogeneity, it does demonstrate the potential for proteolytic triggering of PNT generation from a precursor protein, for example, a fusion protein. When discussing self-assembling protein systems, one must also mention amyloids and amyloid-like fibrils. The self-assembly of amyloid and amyloid-like fibrils has been an area of significant research over the past several years, particularly in the context of neurodegenerative disorders such as Alzheimer's and prion-associated diseases. There are numerous excellent reviews on the structure and assembly of amyloid fibrils, Template-Assembled PNTs An alternative to using self-assembling proteins for PNT generation is to use a template-assisted assembly, which can provide a means of patterned PNT assembly followed by removal of the template layer resulting in free PNTs. In recent study, Tao and colleagues 79 employed a layer-by-layer approach to prepare PNTs composed of two proteins, bovine serum albumin (BSA), and lyophilized hemoglobin from bovine erythrocytes, from glutaraldehyde (GA)-functionalized alumina membranes. The use of the alumina template allowed for uniform PNT length, while pore size and PNT outer diameters could be tailored based upon the alumina support or number of layering steps, respectively. The authors suggested that these template-generated PNTs could be further functionalized, with enzymes or antibodies, to perform targeted catalytic or recognition functions. 79 In another layer-by-layer assembly, human serum albumin (HSA) PNTs were generated by alternating layers of HSA with poly-l-arginine (PLA) from an etched polycarbonate membrane based on the difference in overall net charge between HSA (negative) and PLA (positive). The development of PNT-based biocatalysts has also been explored using layer-by-layer methodologies; the inclusion of an enzyme into the PNT architecture (in whole or in part) provides a specific biocatalytic function. One such example of a biocatalytic PNT is that of the layer-by-layer assembly of cytochrome-C PNTs 81 with either GA or poly-(sodium styrenesulfonate). The cytochrome-C within the PNT architecture retained its native tertiary structure and catalytic activity, suggesting usage of these PNTs in cytochrome-C-specific biocatalytic reactions. Virus-Based Nanotubes Another approach to developing PNTs for nanotechnological/nanomedical applications is the adaptation of viral coat proteins. Many viruses are conveniently rod shaped and their capsids self-assemble from relatively simple protein building blocks, A second tubular nanostucture generated from viral capsid proteins is that of the cowpea chlorotic mottle virus (CCMV). Another virus that has been explored for the development of protein-based bionanowires is the M13 bacteriophage. This process is again a two-step procedure in which the protein scaffold, in this case modified M13 phage coat protein(s), is first assembled and then a conducting molecule selectively aggregates onto the PNT scaffold. A plasmid-driven expression of recombinant M13 coat proteins pIII and pVIII was shown to facilitate the synthesis of gold nanowires; the gold-binding recombinant pIII and pVII proteins were identified through a biopanning process. 91 In a similar study, Nam et al. genetically modified the pVIII coat protein to express four consecutive N-terminal glutamates to serve as a template to produce Co 3 O 4 nanowires 92 CONCLUSION Given the richness of diversity in structures, biocompatibility, and adaptability through genetic engineering, peptide and protein-based nanotubes present unique alternatives to CNT scaffolds for new nanomedical and bionanotechnological applications. The in-built assembly characteristics of flagella, pili, and viral coat proteins are examples of selfassembling nanosystems that show distinct promise in the design and development of bionanosystems for nanomedicine, drug delivery, bionanowiring, etc. In addition, template-assisted PNT generation of layere

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 β-sheet contribute significantly to pilin dimerization. Conversely, the antiparallel β-sheet and αβ loop region exhibit increased flexibility, while the receptor binding domain retains a rigid conformation in the equilibrium state

Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin

Ectromelia virus glutaredoxin has been crystallized in the presence of the reducing agent DTT. A diffraction data set has been collected and processed to 1.8 Å resolution

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 out-weighing 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.BT/Environmental Biotechnolog

Genes responsive to rapamycin and serum deprivation are clustered on chromosomes and undergo reorganization within local chromatin environments.

We previously demonstrated that genome reorganization, through chromosome territory repositioning, occurred concurrently with significant changes in gene expression in normal primary human fibroblasts treated with the drug rapamycin, or stimulated into quiescence. Although these events occurred concomitantly, it is unclear how specific changes in gene expression relate to reorganization of the genome at higher resolution. Using computational analyses, genome organization assays and microscopy, the relationship between chromosome territory positioning and gene expression was investigated. We determined that despite relocation of chromosome territories, there was no substantial bias in the proportion of genes changing expression on any one chromosome, including chromosomes 10 and 18. Computational analyses identified that clusters of serum deprivation and rapamycin-responsive genes along the linear extent of chromosomes. Chromosome conformation capture (3C) analysis demonstrated the strengthening or loss of specific long-range chromatin interactions in response to rapamycin and quiescence induction, including a cluster of genes containing Interleukin-8 and several chemokine genes on chromosome 4. We further observed that the LIF gene, which is highly induced upon rapamycin treatment, strengthened interactions with up- and down-stream intergenic regions. Our findings indicate that the re-positioning of chromosome territories in response to cell stimuli, this does not reflect gene expression changes occurring within physically clustered groups of genes.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

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