Molecular self-assembly and supramolecular chemistry of cyclic peptides

This Review focuses on the establishment and development of self-assemblies governed by the supramolecular interactions between cyclic peptides. The Review first describes the type of cyclic peptides able to assemble into tubular structures to form supramolecular cyclic peptide nanotubes. A range of cyclic peptides have been identified to have such properties, including α-peptides, β-peptides, α,γ-peptides, and peptides based on δ- and ε-amino acids. The Review covers the design and functionalization of these cyclic peptides and expands to a recent advance in the design and application of these materials through their conjugation to polymer chains to generate cyclic peptide–polymer conjugates nanostructures. The Review, then, concentrates on the challenges in characterizing these systems and presents an overview of the various analytical and characterization techniques used to date. This overview concludes with a critical survey of the various applications of the nanomaterials obtained from supramolecular cyclic peptide nanotubes, with a focus on biological and medical applications, ranging from ion channels and membrane insertion to antibacterial materials, anticancer drug delivery, gene delivery, and antiviral applications

Ion Binding and Transport by Synthetic Molecular Assemblies

Self-assembly of small synthetic molecular building blocks has been applied to generate functional structures capable of binding and transporting cations and anions. Major discoveries from the research include a self-assembled ion pair receptor, ligands capable of K+-selective transport through membranes, a compound that forms Cl--selective ion channels in planar and cellular membranes, and a series of efficient chloride transporters. Calix[4]arene-guanosine conjugates cG 2.26 and cG 2.34 are shown to assemble into Na+-templated tubular architectures by 1H NMR, transmission electron microscopy and isothermal titration calorimetry, and to selectively transport K+ over Na+ and Cs+ across liposome membranes by fluorescent assays. The more lipophilic cG 2.34 forms a water-mediated dimer capable of extracting alkali halide salts from water into organic solution. These conclusions are supported by 1H and 23Na NMR, pulsed-field gradient NMR, ion chromatography and circular dichorism spectroscopy. The (cG 2.34)2(H2O)n dimer is held together by an intermolecular hydrogen-bonded guanosine quartet, based on 1D and 2D NMR experiments, and provides a rare example of a self-assembled ion pair receptor. Calix[4]arene tetrabutylamide 3.1 forms voltage-dependent chloride-selective ion channels in planar bilayers and cell membranes based on voltage and patch clamp experiments. Compound 3.1 selectively transports Cl- over HSO4- across liposome membranes, is capable of binding and transporting HCl, and can alter the pH inside liposomes experiencing a chloride gradient, based on fluorescent assays and 1H NMR experiments. X-ray crystal structures of calix[4]arene tetramethylamide 2.30HCl complexes (2.30 is an analogue of 3.1) provides a rationale for how ions are moved across a membrane by 3.1. From a series of linear analogues of 3.1, oligophenoxyacetamide trimer 3.5 was identified as the most potent chloride transporter. Transport of chloride and H+/Cl- pairs was demonstrated by fluorescent assays and 35Cl NMR. Trimer 3.5 also induces a stable potential into liposomes experiencing a transmembrane anionic gradient, an unprecedented function for a synthetic compound. Compounds capable of transporting chloride or H+/Cl- pairs have potential as drugs for the treatment of cystic fibrosis and cancer

Molecular mechanism of action of tyrocidine antimicrobial peptides using NMR spectroscopy and computational techniques

Includes abstract.Includes bibliographical references.The need to come up with new and novel antibiotics that utilize unique mechanisms, to which bacteria cannot generate resistance, was the main motivation of this study. Tyrocidine peptides are non-selective antibiotics that have such properties. However, very limited information is available about their mechanism of action. The aim of this study was to determine the mechanism of action of tyrocidine peptides, tyrocidine A, tyrocidine B and tyrocidine C

Biointerfaces based on the combination of synthetic polymers and biomolecules

Alicat embargament des de la defensa de la tesi fins al 31 de desembre de 2019Premi Extraordinari de Doctorat, promoció 2018-2019. Àmbit d’Enginyeria IndustrialDuring the last decades, research focused on the preparation of highly selective and smart materials has increased considerably. For instances, it has been possible to achieve intelligent drug nano-carriers, biomolecular sensors, platforms to promote cell growth and differentiation among many other striking applications. Two mentionable factors that helped such development are the incorporation of biological moieties onto this interfaces to gain specificity and the combination of more than one material in order to get a synergistic effect between the different components (i.e. conducting polymers suffer from poor mechanical strength, therefore its combination with polyesters can reduce their fragility). This thesis has been devoted to the design and development of high performance polymeric materials for multiple functions related to the biomedical field, such as passive ion transport membranes, drug delivery systems and the addition of selectivity in different surfaces. The work gives special emphasis to the characterization of these platforms, like its surface chemistry, topology, biocompatibility or its mechanical strength. Besides, these systems have been synthetized in a large variety of shapes, from free-standing nanomembranes to polymeric nanoparticles. The Thesis is divided in three blocs: Bloc A encloses all the studies realized for the generation of hybrid nanoperforated membranes in order to achieve controlled ion diffusion. Specifically, an outer membrane protein, Omp2a, was considered for these studies. Primarily, the protein was purified, folded and characterized in an ambient resembling to the one encountered in nature, its mechanical forces and conductivity were analysed. The project was followed by the immobilization of Omp2a into silicon microcantillevers to acquire greater knowledge of its folding and unfolding processes upon thermal stress. Next, artificial polymeric membranes containing nanofeatures were developed with the final purpose to immobilize Omp2a via protein confinement. Then, the conductivity of the membrane with different electrolyte media solutions was tested. Bloc B describes the state-of-the art of drug delivery systems prepared with intrinsically conducting polymers to achieve controlled drug release upon electrical stimuli. Furthermore, two systems based on poly(3,4-ethylenedioxythiophene) (PEDOT) nanoparticles are described. Particularly, curcumin was employed as a model neutral drug and incorporated within the PEDOT nanoparticles. The oxidation state of the PEDOT chains regulated the drug release. Later on, a similar system was generated with polyester microfibers loaded with curcumin and nanoparticles. The driving force for the later drug release was the actuation of the PEDOT nanoparticles. Lastly, Bloc C reports the immobilization of a pentapeptide called CREKA and its analog CR(NMe)EKA onto PEDOT and silicon surfaces. The addition of CREKA favoured the selectivity of those interfaces towards clotted plasma proteins such as fibrin and fibrinogen. PEDOT-peptide material allowed the electrochemical detection of the proteins by an increase in membrane resistance and these interactions were evaluated with microcantilevers by measuring the difference on weight when they were incubated with different protein concentrations. Overall, the compilation of the studies presented in this Thesis offer a comprehensive view on how modifying and generating hybrid materials is possible to optimize and exploit their capabilities for a wide range of applications.Durant les últimes dècades, la recerca centrada en la preparació de materials altament selectius i intel·ligents ha augmentat considerablement. Ha estat possible aconseguir nano-contenidors de fàrmacs, sensors de biomolècules, plataformes per promoure el creixement i la diferenciació cel·lular, entre moltes altres aplicacions interessants. Dos factors destacables que han ajudat aquest desenvolupament són la incorporació de cues biològiques en aquets materials per obtenir especificitat i la combinació de més d'un element per obtenir un efecte sinèrgic entre els diferents components (per exemple els polímers conductors pateixen d’una baixa resistència mecànica, per tant, la seva combinació amb polièsters pot reduir la seva fragilitat però seguir mantenint les seves propietats elèctriques). En resum, aquesta tesi s'ha centrat en el disseny i desenvolupament de materials polimèrics d'alt rendiment per a múltiples funcions relacionades amb el camp biomèdic, com ara membranes passives de transport iònic, sistemes de lliurament de fàrmacs i l'addició de selectivitat envers proteïnes del plasma en diferents superfícies. El treball fa especial èmfasi en la caracterització d'aquestes plataformes, com la seva química superficial, topologia, biocompatibilitat o resistència mecànica. A més, aquests sistemes s'han sintetitzat en una gran varietat de formes, des de films fins a nanopartícules polimèriques. La tesi es divideix en tres blocs: El bloc A inclou tots els estudis realitzats per a la generació de membranes híbrides nanoperforades amb la finalitat d’aconseguir una difusió controlada de ions. Concretament en aquests estudis es va emprar una proteïna transmembrana anomenada Omp2a. La primera etapa del treball es centra en la purificació, plegament i caracterització de la proteïna en un ambient similar al que es troba originàriament. A més a més, es van analitzar les seves forces mecàniques i de conductivitat. Seguidament, es va procedir a la immobilització d'Omp2a en microcantillevers de silici per adquirir un major coneixement sobre els seus processos de plegament i desplegament depenent de l'estrès tèrmic. Finalment, es van desenvolupar membranes polimèriques artificials amb nanoperforacions amb l'objectiu d'immobilitzar Omp2a a través del confinament de la proteïna en aquests porus. El Bloc B descriu l'estat de l’art dels sistemes d’alliberament controlat de fàrmacs, preparats amb polímers intrínsecament conductors, depenent d’estímuls elèctrics. En aquest apartat, es descriuen dos sistemes basats en nanopartícules de poli(3,4-etilendioxitiofé) (PEDOT). En el primer cas, l'estat d'oxidació de les cadenes PEDOT és el responsable de regular l'alliberament del medicament. En canvi, en el segon, on es va generar un sistema similar amb microfibres de polièster carregades de droga i nanopartícules per separat, la força motriu de l'alliberament del fàrmac és el moviment d’expansió i contracció de les nanopartícules PEDOT. Finalment, el Bloc C informa de la immobilització d'un pentapèptid anomenat CREKA i el seu anàleg CR(NMe)EKA en films de PEDOT i superfícies de silici. La incorporació de CREKA afavoreix la selectivitat d'aquestes interfícies cap a les proteïnes de coagulació del plasma com la fibrina i el fibrinogen. El material pèptid-PEDOT va permetre la detecció electroquímica de les proteïnes mitjançant un augment de la resistència a la membrana i aquestes interaccions van ser avaluades amb microcantilevers, concretament, mesurant la diferència de pes quan es van incubar amb diferents concentracions de proteïnes. En general, la recopilació d’aquets estudis ofereix una visió completa sobre com modificant i generant materials híbrids és possible optimitzar i explotar les seves capacitats particulars per a una àmplia gamma d'aplicacions.Award-winningPostprint (published version

Toward biologically realistic computational membrane protein structure prediction and design

Membrane proteins function as gates and checkpoints that control the transit of molecules and information across the lipid bilayer. Understanding their structures will provide mechanistic insights in how to keep cells healthy and defend against disease. However, experimental difficulties have slowed the progress of structure determination. Previous work has demonstrated the promise of computational modeling for elucidating membrane protein structures. A remaining challenge is to model proteins coupled with the heterogeneous cell membrane environment. In the first half of this dissertation, I detail the development, testing and integration of a biologically realistic implicit lipid bilayer model in Rosetta. First, I describe the initial iteration of the implicit model that captures the anisotropic structure, shape of water-filled pores, and nanoscale dimensions of membranes with different lipid compositions. Second, I explain my approach to energy function benchmarking and optimization given the challenge of sparse and low-quality experimental data. Third, I outline the second generation that incorporates a new electrostatics and pH model. All of these developments have advanced the accuracy of Rosetta membrane protein structure prediction and design. In the second half of this dissertation, I investigate three challenging biological and engineering applications involving membrane proteins. In the first application, I examine mutation-induced stability changes in the integral membrane zinc metalloprotease ZMPSTE24: a protein with a large voluminous chamber that is not captured by current implicit models. In the second application, I model interactions between the SERCA2a calcium pump and the regulatory transmembrane protein phospholamban: a key membrane protein-protein interaction implicated in the heart’s response to adrenaline. Finally, I explore the challenge of membrane protein design to engineer a self-assembling transmembrane protein pore for nanotechnology applications. These applications highlight the next steps required to improve computational membrane protein modeling tools. Taken together, my work in both methods development and applications has advanced our understanding and ability to model and design membrane protein structures

New Platforms for Optical Biosensing

Physicochemical experimental techniques combined with the specificity of a biological recognition system have resulted in a variety of new analytical devices known as biosensors. Biosensors are under intensive development worldwide because they have many potential applications, e.g. in the fields of clinical diagnostics, food analysis, and environmental monitoring. Much effort is spent on the development of highly sensitive sensor platforms to study interactions on the molecular scale. In the first part, this thesis focuses on exploiting the biosensing application of nanoporous gold (NPG) membranes. NPG with randomly distributed nanopores (pore sizes less than 50 nm) will be discussed here. The NPG membrane shows unique plasmonic features, i.e. it supports both propagating and localized surface plasmon resonance modes (p SPR and l-SPR, respectively), both offering sensitive probing of the local refractive index variation on/in NPG. Surface refractive index sensors have an inherent advantage over fluorescence optical biosensors that require a chromophoric group or other luminescence label to transduce the binding event. In the second part, gold/silica composite inverse opals with macroporous structures were investigated with bio- or chemical sensing applications in mind. These samples combined the advantages of a larger available gold surface area with a regular and highly ordered grating structure. The signal of the plasmon was less noisy in these ordered substrate structures compared to the random pore structures of the NPG samples. In the third part of the thesis, surface plasmon resonance (SPR) spectroscopy was applied to probe the protein-protein interaction of the calcium binding protein centrin with the heterotrimeric G-protein transducin on a newly designed sensor platform. SPR spectroscopy was intended to elucidate how the binding of centrin to transducin is regulated towards understanding centrin functions in photoreceptor cells.Physikochemische instrumentelle Techniken, die mit der Spezifität eines biologischen Erkennungssystems kombiniert sind, resultierten in unzähligen neuen analytischen Geräten bekannt als Biosensoren. In die Entwicklung von Biosensoren wird weltweit viel investiert angesichts der zahlreichen potentiellen Anwendungen, wie z.B. in der klinischen Diagnostik, der Nahrungsmittelanalyse und zur Umweltüberwachung. Hochempfindliche Sensor-Plattfor-men werden benötigt, um Wechselwirkungen auf molekularer Ebene zu studieren. Im ersten Teil der Doktorarbeit werden nanoporöse Gold (NPG)-Membranen im bezug auf ihre biosensorische Anwendung untersucht. NPG Proben mit einer willkürlichen Porengrößenverteilung (Poren von weniger als 50 nm) werden hierzu erforscht. Die NPG Membranen zeigen einzigartige plasmonische Eigenschaften, d.h. propagierende und lokalisierte Oberflächenplasmonresonanzmodi (p-SPR bzw. l-SPR) können gleichzeitig angeregt werden. Beide Moden ermöglichen eine sensitive Detektion der lokalen Brechungsindexveränderung an/im nanoporösen Gold Substrat. Der große Vorteil der Brechungsindexsensoren im Vergleich zu fluoreszenz-optischen Biosensoren besteht darin, daß keine chromophore Gruppe oder Lumineszenzmarkierung zur Detektion benötigt wird. Im zweiten Teil der Arbeit wurden macroporöse, aus Gold und Silica zusammengesetzte inverse Opale auf ihre bio- bzw. chemischen Sensorfähigkeiten hin analysiert. Diese Substrate kombinieren den Vorzug einer großen verfügbaren Oberfläche mit einer hoch geordneten Gitterstruktur. Das Plasmonensignal ist bei einer geordneten Substratstruktur weniger verrauscht als es bei der willkürlichen Anordnung der Poren im NPG der Fall ist. Im dritten Teil der Doktorarbeit wird die Oberfächenplasmonenresonanz (SPR) Spektroskopie angewendet, um die Protein-Protein Wechselwirkung zwischen dem Calcium bindenden Protein Centrin und dem heterotrimeren G-Protein Transducin zu erforschen. Dazu wurde eine neue Sensorplattform entwickelt. Die SPR Spektroskopie sollte aufklären, wie die Bindung des Centrins zum Transducin reguliert wird und zum besseren Verständnis der Centrinfunktionen in den Photorezeptorzellen beitragen

Une membrane souple à base de polypyrrole renforcée et son utilisation pour délivrer des stimulations électriques aux kératinocytes de peau humaine

La stimulation électrique (SE) semble favoriser la cicatrisation des plaies par ses effets sur les fibroblastes. Cependant, son interaction avec les kératinocytes n'a pas été bien établie. Le polypyrrole (PPy) en tant que biomatériau conducteur est un excellent candidat pour délivrer les SE aux cellules, ce qui devient plus évident avec le développement de la nouvelle membrane souple à base de PPy. Cependant, les faibles propriétés mécaniques limitent l'utilisation de cette membrane. La présente étude visait à améliorer la résistance mécanique de la membrane à base de PPy et étudier les comportements cellulaires et moléculaires des kératinocytes après exposition à des SE via cette nouvelle membrane PPy. Premièrement, la membrane souple à base de PPy a été renforcée par électrofilage, de manière synergique, avec des fibres de polyuréthane (PU) et de polylactide (PLLA). Des tests mécaniques ont confirmé que la résistance à la traction de la membrane a été considérablement augmentée. Ensuite, les kératinocytes ont été cultivés sur la membrane PPy renforcée, puis stimulés par des intensités électriques de 100 ou 200 mV mm⁻¹ pendant 6 ou 24 heures. Les cellules stimulées présentaient une capacité proliférative considérablement accrue. Les sécrétions d'IL-6, IL-1α, IL-8, GROα, FGF2 et VEGF-A ont également augmenté. Fait intéressant, l'SE de 24 heures a induit une « mémoire de stimulation » car les cellules stimulées ont montré une augmentation significative de formation de colonies (CFE) après 6 jours après l'exposition à la stimulation électrique. De plus, l'expression des kératines 5, 14 et 10/13 était significativement augmentée par la SE. La SE a augmenté l'expression de la phosphorylation des kinases ERK1/2. L'expression des protéines des kératinocytes de la peau humaine peut être activée par des stimulations électriques appropriées pour favoriser la cicatrisation des plaies cutanées. La membrane PPy souple renforcée peut servir de pansement conducteur pour faciliter l'exposition de la plaie à une stimulation électrique pour favoriser sa cicatrisation.Keratinocytes as the principal skin cell type play a major role in wound closure. In the meantime, electrical stimulation (ES) has been found effective in promoting wound healing. However, the role of ES on keratinocytes has not been well established. Polypyrrole (PPy), especially the recently developed soft PPy membrane, is an electrically conductive biomaterial and a good candidate to deliver ES to cells. However, the weak mechanical strength of the soft PPy membrane has limited its practical use. The present work was to enhance the mechanical strength of this soft PPy membrane and to investigate the cellular and molecular behaviors of the keratinocytes underwent ES via this novel PPy membrane. Firstly, the soft PPy membrane was synergically reinforced with polyurethane (PU) and poly (L-lactic acid) (PLLA) fibers through electrospinning technology. Mechanical tests confirmed the significantly increased tensile strength, which rendered the originally fragile PPy membrane strong enough to stand ordinary manipulations without compromising its electrical properties. Afterwards, HaCaT keratinocytes were cultured on the PU/PLLA reinforced PPy membranes under electrical intensities of 100 and 200 mV mm⁻¹ for 6 or 24 hr. The electrically stimulated cells exhibited a considerably increased proliferative ability. Meanwhile, secretions of the IL-6, IL-1α, IL-8, GROα, FGF2 and VEGF-A increased as well. Interestingly, the 24 hr ES induced a "stimulus memory" by showing a significant rise in colony forming efficiency (CFE) 6 days post-ES. Additionally, the expressions of keratin 5, keratin 14, keratin 10 and keratin 13 were significantly modulated by ES. Finally, the phosphorylation of ERK1/2 kinases was regulated by ES. The overall results demonstrated that the proliferation, differentiation, and protein expression of human skin keratinocytes can be activated through appropriate ES to benefit skin wound healing. Moreover, the PU/PLLA reinforced soft PPy membrane may server as a conductive wound dressing to facilitate ES to wound