Structural analysis of cross α-helical nanotubes provides insight into the designability of filamentous peptide nanomaterials

The exquisite structure-function correlations observed in filamentous protein assemblies provide a paradigm for the design of synthetic peptide-based nanomaterials. However, the plasticity of quaternary structure in sequence-space and the lability of helical symmetry present significant challenges to the de novo design and structural analysis of such filaments. Here, we describe a rational approach to design self-assembling peptide nanotubes based on controlling lateral interactions between protofilaments having an unusual cross-α supramolecular architecture. Near-atomic resolution cryo-EM structural analysis of seven designed nanotubes provides insight into the designability of interfaces within these synthetic peptide assemblies and identifies a non-native structural interaction based on a pair of arginine residues. This arginine clasp motif can robustly mediate cohesive interactions between protofilaments within the cross-α nanotubes. The structure of the resultant assemblies can be controlled through the sequence and length of the peptide subunits, which generates synthetic peptide filaments of similar dimensions to flagella and pili

Application of molecular simulation techniques to the design of nanosystems

Nanotechnology is a multidisciplinary branch of science and technology that involves a widerange of different fields such as chemistry, materials science, physics or chemical engineeringwhose goal is the production of new functional materials and devicesthrough the control of their organization at the atomic and molecular scale.Nanotechnology has jumped from research laboratories to our daily life and today all theprogresses made in this field have been translated into direct applications in different fields being electronics and computer science and biomedicine, where the most striking advances have beendone.What differences nanotechnology from traditional chemistry and physics can be summarized inthree points: (i) Analysis and control of the matterat the atomic and molecular level focusing in individual atoms; (ii) the appearance of novel physical properties because of the nanoscopicdimensions; (iii) the possibility of generating new complex functional systems with novelproperties.Modeling and theory are becoming vital to designing and improving nanodevices. The intrinsicnature of nano and supramolecular scale that involves tens, hundreds and thousands of atomsmakes computational chemistry the perfect ally to design new devices and predict their properties. Computational chemistry provides the perfect tools to describe the electronic structureand the dynamic behavior, as well as the properties derived from them, through quantummechanics and classical mechanics formalisms.The suitability of such techniques in the design and improvement of nanodevices as well as theprediction of their properties is clearly proven throughout the four blocks in which this thesis isdivided:· Nanotubes based on natural peptide sequencesNanotubes have gained extensive interest because of their applicability in different fieldsranging from medicine to electronics. Among nanotubes, those based on natural peptidesequences taken from some natural proteins with a tubular or fibrillar motif are gaining abroad attention because of their high biocompatibility, the possibility of adding functionalitiesby tuning them and their potentiality to self-assemble. The enhancement of the ability to retain the tubular geometry of such structures can be achieved by substituting targeted amino acids located in the more flexible parts of the nanoconstruct by synthetic amino acids withlow conformational flexibility providing a larger rigidity to the overall structure.· Dendronized polymersDendronized polymers are a specific kind of macromolecule structure that consists of a linearpolymeric backbone where dendritic units are attached regularly leading to a highly branchedthree-dimensional architecture. This fact provides dendronized polymers the peculiarity of the coexistence within the same macromolecule of three topological regions: (i) the internalbackbone; (ii) the dendron region around the backbone and (iii) the external surface. Thesemolecules have a wide range of applications in different fields such as biomedical engineering, host-guest chemistry or catalysis.· Theoretical study of ð-conjugated systemsConducting polymers are polymers bearing a characteristic polyconjugated nature which makethem electronic conductors. In particular thiophene-based conducting polymers have been widely studied because of their electric and nonlinear optical properties, excellent environmentalstability and relatively low cost of production. Due to the crucial role played by the electronicstructure of these systems in their relevant properties, a good knowledge of it is a key factor todesign and improve new conducting polymers. To achieve this goal QM calculations suitperfectly to get accurate estimates of such properties.· Molecular actuators and sensors based on conducting polymersBoth experimental and computational research in nanoactuators and nanosensors are widelyreported in the literature. Among them, those based in conducting polymers are flourishingbecause of their great transport properties, electrical conductivity or rate of energy migrationwhich provide amplified sensitivity in nanosensors and a rapid response in nanoactuators. In thissense electron-rich thiophene-based oligomers and polymers combined with versatilecalix[4]arenes units are presented in the present thesis. Calix[4]arenes are synthetic macrocyclic molecules consisting of four phenol or anisole rings connected via methylene bridges that canhost different guest molecules leading to conformational rearrangement of the whole device making it useful to be employed as a sensor or actuator

Multidomain Peptides: Sequence-Nanostructure Relationships and Biological Applications

Peptides are materials that, as a result of their polymeric nature, possess enormous versatility and customizability. Multidomain peptides are a class of peptides that selfassemble to form stable, cytocompatible hydro gels. They have an ABA block motif, in which the A block is composed of charged amino acids, such as lysine, and the B block consists of alternating hydrophilic and hydrophobic amino acids, such as glutamine and leucine. The B block forms a facial amphiphile that drives self-assembly. The charged A blocks simultaneously limit self-assembly and improve solubility. Self-assembly is triggered by charge screening of these charged amino acids, enabling the formation of ~sheet fibers. The development of an extended nanofiber network can result in the formation of a hydrogel. Systematic modifications to both the A and B blocks were investigated, and it was found that sequence modifications have a large impact on peptide nanostructure and hydrogel rheology. The first modification examined is the substitution of amino acids within the hydrophilic positions of the B block. The second set of modifications investigated was the incorporation of aromatic amino acids in the B block. Finally, the charged block was varied to generate different net charges on the peptides, a change which impacted the ability to use these peptides in cell culture. Two applications of multi domain peptide nanofibers are explored, the first of which is the delivery of novel therapies in vivo. One multidomain peptide is able to form hydrogels that undergo shear-thinning and rapid recovery. This gel can be loaded with cytokines and growth factors that have been secreted by embryonic stem cells, and these molecules can be subsequently released in a therapeutic setting. Another application for multidomain peptide is their use as biocompatible surfactants. Single-walled carbon nanotubes have been widely investigated for their unique optical and electrical properties, but their solubility in aqueous systems has been a challenge. Multidomain peptides solubilize carbon nanotubes, are less cytotoxic than detergents such as SDBS, and preserve the ability of carbon nanotubes to fluoresce. Some of these peptides are also compatible with cell culture, allowing the delivery of single-walled carbon nanotubes to cells

Exploration of Peptide-Thiophene Hybrids as Self-Assembling Conductive Hydrogels

Here, we have taken a bottom-up approach to confer multidimensional structure to conductive polymers by attaching thiophene monomers to peptides predicted to self-assemble into a biomimetic, fibrous nanostructure. A library of 12 peptides containing covalently attached thiophene-based monomers was synthesized. Peptide sequences that resulted in self-assembly and hydrogel formation in aqueous media were identified and the physical and electrical properties were characterized. The resulting hybrid materials have conductivities in the range of 10-2-10-3 S/cm, and possess moduli in the range of several tissue types, making them potential candidates for use in biomedical electronic applications

Self-Assembling Peptide-Carbon Nanotube Dispersions and Hydrogels for Tissue Engineering and Biosensor Applications

Carbon nanotubes (CNTs) are attractive functional materials for use in a broad range of fields due to their unique mechanical and electrical properties. However, their hydrophobic nature is a major problem for some of these applications. Several approaches such as dispersing them in organic solutions and covalently or non-covalently modifying them have been developed to make CNTs usable for desired applications. Since organic solutions can be problematic for bio-applications and covalent modification can introduce defects into the CNT structure (responsible for its unique properties), the approach of making non-covalent modification is more promising. Different types of polymers and surfactants have been used so far in this way. In the past two decades, self-assembling peptides have emerged as promising functional nanomaterials capable of use for different bio-applications. Employing biocompatible self-assembling peptides for CNT dispersion not only removes the first obstacle to use CNTs in solution, but also can result in a new class of hybrid nanomaterials benefitting from the synergistic effects of peptides and CNTs. To the best of our knowledge, this is the first work reporting the dispersion of CNTs using β-sheet-forming self-assembling ionic-complementary peptides. Also this is the first study on the application of peptide-CNT hybrid dispersions and hydrogels for biosensor and tissue engineering applications. This thesis focuses on the modification of CNTs with self-assembling peptides, characterization of the resulting hybrid dispersions and their application for biosensor development and scaffolding for tissue engineering and cancer spheroid studies. In particular, the study includes the following topics: (i) characterization of the dispersions of multi-walled carbon nanotubes (MWNTs) modified with EFK16-II peptide, (ii) AFM characterization of dispersions of single-walled carbon nanotubes (SWNTs) modified with EFK8 peptide, (iii) formation of hybrid EFK8-SWNT hydrogels, (iv) application of the hybrid EFK8-SWNT dispersion in electrode modification and design of a hemoglobin biosensor, and (v) application of the EFK8 and EFK8-SWNT hydrogels as scaffolds for tissue engineering and 3D cancer cell spheroid formation. First, we have shown that by non-covalently modifying MWNTs with the ionic-complementary peptide EFK16-II, very stable dispersions of MWNTs can be formed due to the electrostatic repulsion between self-assembled peptides on the MWNTs. Zeta potential and DLS measurements indicated that as the pH diverges from the isoelectric point of ~ 6.7 for EFK16-II, the repulsion between the particles increases and their resulting sizes decrease. AFM, SEM and TEM studies revealed a uniform distribution of individual modified MWNTs. Finally, tissue culture plates treated with these hybrid dispersions were found to have enough biocompatibility for cell attachment and growth. In the next step, the peptide EFK8, a shorter version of EFK16-II, was used to disperse SWNTs in water. Scanning probe microscopy (SPM) techniques based on nano-mechanical measurements and electric force microscopy (EFM) were used to more closely examine the structure formed between nanotubes and peptides. The SPM images reveal a structure consistent with EFK8 fibers wrapping around SWNTs and rendering their outer surfaces hydrophilic which enables their dispersion in water. Also it was shown that the hybrid dispersions can form uniform composite EFK8-SWNT hydrogels upon adding solutions containing ≥1mM monovalent salts. In the third part of the study, EFK8 and EFK8-SWNT hybrid hydrogels were prepared and used to culture NIH-3T3 fibroblast and A549 lung cancer cells. The effect of the presence of SWNTs in the peptide hydrogel on NIH-3T3 cells behavior cultured on hydrogels was first investigated. Inverted light and confocal microscopy images showed that although cells grow they tend to maintain spherical morphology and form colonies on the EFK8 hydrogel. The presence of SWNTs significantly improved cell behavior so that they exhibited a stretched morphology, spread individually and homogeneously over the surface and proliferated faster. In addition, the cells were observed to migrate into the hydrogel after being seeded on top of the hydrogel. Micro-indentation tests showed that increasing EFK8 solution concentration led to an increase in the hydrogel compressive modulus, whereas the presence of SWNTs did not have any effect in this case. So the beneficial effect of SWNTs on cell behavior cannot be attributed to mechanical property modification and is probably due to their providing locations for cell anchorage that facilitate attachment, spreading and migration. The cells encapsulated in both hydrogels showed the same behavior as in 2D environments (i.e., forming colonies on EFK8 and spreading individually on the hybrid hydrogel). In the second part of this study, the potential of EFK8 hydrogels for spheroid formation of cancer cells was explored. These cancer cell spheroids can be used as models for real tumors, to carry out drug screening in 3D cell cultures and to investigate the effect of the microenvironment on tumor progression and metastasis. It was observed that cells formed spheroids on EFK8 hydrogels at normal peptide concentrations, but exhibited a more stretched morphology and migratory phenotype when seeded on the stiffer hydrogel. The cells also adopted a stretched morphology with higher migration when seeded on the EFK8-SWNT hydrogels. Again this behavior can be attributed to the effect of SWNTs to facilitate cell adhesion and migration. This effect can be used to study another effect of the microenvironment, namely cell-binding motifs, on tumor progression and metastasis. In the last step of the study, the application of the hybrid EFK8-SWNT dispersion was investigated for immobilization and direct electrochemistry of hemoglobin (Hb) on a glassy carbon electrode (GCE) as well as the efficacy of this platform for making a hydrogen peroxide (H2O2) biosensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments showed that the presence of SWNTs in the modifying peptide layer on GCE significantly enhances the electrochemical response of the electrode. Furthermore, this response was further increased as more layers of the EFK8-SWNT dispersion were applied. The next step was to immobilize hemoglobin on the electrode by a casting method. The effectiveness of this approach was confirmed by CV and EIS experiments which showed that immobilized Hb retained its bio-catalytic activity for Fe ions in Hb chains and could form the basis of a mediatorless H2O2 biosensor. Overall, by expanding the functionalities of both CNTs and self-assembling peptides, this work has introduced a new hybrid nanomaterial for bio-applications, especially biosensors, 3D cell cultures and tissue engineering.4 month

Insights into the protein interactions with graphitic nanomaterials using computational modelling techniques

In this thesis computational modelling techniques were employed to investigate the behaviour of an amyloidogenic protein in the presence of dimensionally different carbonaceous nanomaterials. Experimental studies have demonstrated that nanoparticles can affect the rate of protein self-assembly, possibly interfering with the development of protein misfolding diseases such as Alzheimer’s, Parkinson’s and prion disease caused by aggregation and fibril formation of amyloid-prone proteins. The computational techniques used in this thesis include large-scale density functional theory calculations and classical molecular dynamics simulations and their derivative methods such as umbrella sampling and replica exchange methods. The behaviour, in solution and various fibril inhibiting and fibril favouring environments, of the amyloidogenic protein apolipoprotein C-II (ApoC-II) and its peptides derivatives are discussed in detail. Additionally the graphitic nanomaterials (C60, single walled carbon nanotube and graphene) used throughout this thesis are also described. Initially the effect of the different carbon nanomaterials on the structure, dynamics and binding of the monomeric peptide apoC-II(60-70), all of which can influence its fibril formation capacity, were studied and compared to results obtained from previously characterised peptide behaviour in solution. A combination of computational methods, including large-scale electronic structure calculations and classical all-atom molecular dynamics was utilised. Understanding the effect nanomaterials may have on the early stages of fibril formation and in particular the small oligomeric species that drive the initial fibrillation behaviour is important in identifying how they can either inhibit or promote fibril growth. The adsorption and desorption mechanism of two preformed oligomeric composits of apoC-II(60-70) peptide (dimer and tetramer) were investigated. The adsorption mechanism of the apoC-II(60-70) dimer and tetramer to each nanomaterial was investigated and the results were used to determine if there existed a favourable adsorption mechanism that could impact on the fibrillation ability of the oligomeric peptides. The advanced sampling method known as replica exchange with solute tempering (REST) was applied to investigate the stability and interactions of the apoC-II(60-70) dimer and tetramer while adsorbed to the carbonaceous nanomaterials. The results were used to rationalise how nanomaterial curvature, fibril seed size and peptide - nanomaterial binding energy impact the oligomers stability and thus fibrillation ability. The aggregate dynamics and structure of the full length preformed apoC-II tetramer in the presence the different carbonaceous nanomaterials was also explored. The conformational stability and structural dynamics of the apoC-II tetramer while adsorbed to each surface was determined. The results were compared to those obtained throughout the thesis of the peptide derivative apoC-II(60-70) and its oligomers in order to extrapolate the potential effect that these nanomaterials may have on influencing the fibril stability of the full length apoC-II tetramer. To conclude a summary of the overall findings of the thesis and how they may impact the wider research community is presented. Additionally, possible future research into the effect that other graphitic nanomaterials, including graphene-oxide, may have on fibrillation. Another amyloidogenic peptide, amylin, is also discussed as a future target for combating type II diabetes with nano therapeutics

Engineered proteins as molecular templates for functional materials.

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Exploration of carbon nanotube composites and piezoelectric materials for implantable devices

This thesis describes an exploration of carbon nanotube (CNT) nanocomposites for application in implantable medical devices. The focus here is on materials and structures of interest as components of devices incorporating electrodes. Electrodes for implantable devices are commonly required to interface between an electrical system, where the charge carriers are electrons presented through a metal, and human tissue, where the charge carriers are ions as well as electrons not in a metal. These interfaces are found to be prone to issues such as fibrosis and rejection. The properties of carbon nanomaterials, piezoelectric peptides/polymers and their composites suggest them as promising candidate materials that could resolve these issues. The superior conductivity, mechanical properties and chemical stability of carbon nanotubes have been explored in recent years for potential application in biomedical sensors and devices. This work has explored piezoelectric materials, carbon nanotubes, polymers and nanocomposites of these as potential components of implantable devices. Diphenylalanine is a chiral, amphiphilic dipeptide molecule which has the ability to self-assemble into piezoelectric microtubules. The self-assembly process of diphenylalanine microtubules has been explored and its properties have been compared to the properties of poly[vinylidenefluoride-co-trifluoroethylene] (P[VDF-TrFE]) electrospun nanofibres. Later parts of this work considered the deposition of electrodes by printing. The development of CNT-polymer nanocomposites as printable inks for the fabrication of electrodes was explored. The structure and properties of the piezoelectric nano/ micro-materials, CNT-peptide complex and conductive CNT-polymer printable inks were characterised by a range of microscopic and spectroscopic techniques. The viability of neural cells on the developed functional materials and electrodes were tested by metabolic activity measurements and immunochemical staining microscopy. A CNT-polymer ink demonstrated good conductivity and dimensional stability when printed by 3D printer. Good biocompatibility of all the functional materials developed have been demonstrated in vitro, showing promise for further development of soft electrodes and applications in nanostructure piezoelectric sensors and implantable devices