Chirality Effects on Peptide Self-Assembly Unraveled from Molecules to Materials

Self-assembling short peptides are attractive minimal systems for mimicking the constituents of living systems and building (bio)materials. The combination of both D- and L-amino acids into heterochiral sequences is a versatile strategy for building durable supramolecular architectures, especially when their homochiral analogs do not self-assemble. The reasons for this divergent behavior have remained obscure until now. Here, we elucidate how and why homochiral and heterochiral peptides behave differently. We identify a key spectroscopy signature and its corresponding molecular conformation, whereby an amphiphilic structure is uniquely enabled by the peptide stereochemistry. Importantly, we unravel the self-assembly process as a continuum from the conformation of single molecules to their organization into nano- and microstructures and through to macroscopic hydrogels, which are probed for cytotoxicity in fibroblast cell culture. In this way, (bio)material properties at the macro-scale can be linked to the chemical structure of their building blocks at the angstrom scale. Nature makes pervasive use of homochirality (e.g., D-sugars and L-peptides) to assemble biomolecules, whose interactions determine life processes. D-amino acids rarely occur, and their effects are not yet completely understood. For a long time, structural complexity (e.g., polypeptides and constrained molecules) was considered a requirement for achieving defined conformations that ultimately allow biomolecule recognition and function. Here, we detail how minimalist building blocks can adopt conformations with a characteristic spectroscopic signature, whereby substitution of just one L-amino acid for its D mirror image leads to a divergent path for assembly in water. Subtle molecular variations are amplified through increasing size scale all the way to macroscopic differences that are visible to the eye. Ultimately, the design of heterochiral (bio)molecules thus provides an alternative approach to shed new light on the supramolecular interactions that define life as we know it. This work explains why and how heterochiral and homochiral tripeptides differ in their assembly in water. A characteristic spectroscopic signature is assigned to molecular conformation. We monitor the process as a continuum from the molecular scale to the macroscopic biomaterials so that the final properties are linked to chemical structure of the building blocks. This work lays the foundation for the design of supramolecular hydrogel biomaterials based on short sequences of hydrophobic D- and L-amino acids

Characterization of two membrane proteins: a P-IV ATPase from yeast and a bacterial polymerase

Lo scopo di questo lavoro \ue8 la caratterizzazione di due proteine di membrana coinvolte in processi fisiologici per il mantenimento delle caratteristiche fondamentali delle membrane cellulari. La prima proteina \ue8 Neo1 da Saccharomyces cerevisiae, appartenente alla classe delle P-IV ATPasi, proteine essenziali nel mantenimento dell\u2019asimmetria della membrana. A questo scopo, le P-IV ATPasi trasferiscono fosfolipidi dalla parte esterna delle membrane a quella interna. Nell\u2019uomo, malfunzionamenti di queste proteine sono connessi a malattie come malattie del fegato, Alzheimer, obesit\ue0 e diabete di tipo 2. Visto il ruolo cruciale di queste proteine, la delucidazione del loro meccanismo di azione \ue8 di fondamentale importanza. Neo1 da S. cerevisiae, omologa ad una sottoclasse di proteine umane, \ue8 stata studiata sfruttando l\u2019alta omologia tra P-IV ATPasi umane e di lievito. Neo1 \ue8 l\u2019unica P-IV ATPasi di S. cerevisiae essenziale per la sopravvivenza cellulare, ma i suoi substrati sono tuttora sconosciuti. La proteina selezionata \ue8 stata espressa come chimera con un Green Fluorescent Protein (GFP)/istidine tandem tag. Tra i vari detergenti testati per la solubilizzazione di Neo1, solo in LMNG \ue8 stato possibile ottenere una proteina purificata e stabile. Con analisi al dicroismo circolare una temperatura di fusione tra 40-50\ub0C\ue8 stata rilevata. Saggi di attivit\ue0 sono stati effettuati sulla proteina in presenza di diversi lipidi per identificare il substrato di Neo1. Mutanti della proteina sono stati espressi per testare il ruolo di specifici residui coinvolti nell\u2019attivit\ue0 della proteina. Nelle immagini EM a colorazione negativa ad alto ingrandimento di Neo1 si vede un campione senza aggregazione. Questo risultato rappresenta pertanto un eccellente punto di partenza per analisi future. La seconda proteina \ue8 Wzy da Pseudomonas aeruginosa, batterio connesso ad infezioni ospedaliere spesso trovato nei polmoni di pazienti affetti da fibrosi cistica. Nei batteri, un denso muro di polisaccaridi ricopre la membrana cellulare ed \ue8 coinvolto in processi cruciali durante l\u2019infezione batterica. Wzy formara nuovi legami glicosidici tra corte unit\ue0 oligosaccaridiche, contribuendo cos\uec alla formazione del lipopolisaccaride (LPS) che compone il muro polisaccaridico nei batteri gram negativi. Poich\ue9 l\u2019LPS \ue8 altamente specifico per ogni batterio e ceppo, inibitori della sua via di biosintesi dovrebbero essere selettivi contro un batterio specifico. Pertanto la comprensione della struttura di Wzy rappresenterebbe un passo avanti per la creazione di nuovi antibiotici contro P. aeruginosa. In questo studio, Wzy \ue8 stata clonata e overespressa in E. coli come chimera assieme alla GFP e un tag di istidine. La proteina \ue8 stata solubilizzata in diversi detergenti e purificata con metodi cromatografici. Dai risultati della purificazione \ue8 stato possibile identificare i detergenti migliori per ottenere una proteina pura e stabile perle analisi successive. La stabilit\ue0 \ue8 stata testata con dicroismo circolare, spettroscopia Raman e spettroscopia di fluorescenza. Sia in CD che in spettroscopia di fluorescenza, \ue8 stato determinato un punto di fusione di circa 58\ub0 C. Diversi cristalli sono stati ottenuti da prove di cristallizzazione. Da esperimenti di difrazione alla linea XRD2 del Sincrotrone Elettra su questi cristalli, dati a circa 6 \uc5 di risoluzione sono stati ottenuti, ma la soluzione della struttura ha rivelato che questi cristalli sono formati da un contaminante presente nel campione, la Citocromo O Ubiquinolo Ossidasi. Esperimenti successivi hanno suggerito una stretta interazione tra Wzy e la Citocromo O Ubiquinolo Ossidasi, con un effetto di stabilizzazionesu Wzy. Sul campione di proteina e contaminante sono stati effettuate analisi EM a colorazione negativa, confermando che il campione \ue8 monodisperso e omogeneo, essendo pertanto utilizzabile per future analisi EM.The cellular membrane is a physical barrier that divides the interior of the cell, the cytosol, from the extracellular environment. In this work I focused on the characterization of two membrane proteins involved in physiological processes that maintain the crucial features of cellular membranes. The first protein is Neo1 of Saccharomyces cerevisiae, belonging to the P-IV ATPase family and essential to maintain membrane asymmetry. To this aim, P-IV ATPases translocate phospholipids from the outer to the inner leaflets of the membranes. In humans, malfunction of these proteins are related to pathologies like liver diseases, Alzheimer\u2019s, obesity and type 2 diabetes. Considering the key role of these proteins, the clarification of their mechanism of action is of utmost importance. Neo1 from S. cerevisiae, homologous of a P-IV ATPase subclass in human, was studied Exploiting the high homology between human and yeast P-IV ATPases. Neo1 is the only S. cerevisiae P-IV ATPase essential for cell survival, but its substrates are still a matter of debate. The target protein was expressed as a chimera with a green fluorescence protein (GFP)/histidine tandem tag. For membrane proteins, the choice of the proper solubilization detergent is a crucial step for their characterization. Among the various detergents tested for Neo1 solubilization, a pure and stable protein could only be obtained in LMNG. Circular dichroism analysis of the sample indicated a melting temperature in the range 40-50\ub0 C. Activity assays were performed on the purified protein in presence of different lipids in order to identify Neo1 substrate. Mutants of the protein were expressed to test the role of specific residues in protein activity. Negative staining EM images at high magnification showed a homogeneous sample without aggregation, representing an excellent starting point for further analysis. The second protein is the polymerase Wzy from Pseudomonas aeruginosa. P. aeruginosa is a pathogen related to hospital-acquired infections and often found in the lungs of cystic fibrosis patients. In bacteria, a thick polysaccharide wall covers the cell membrane and is involved in crucial infection steps. The glycosyltransferase Wzy is a fundamental part of the pathway for the production of the lipopolysaccharide (LPS) on the outer membrane of gram-negative bacteria and its role is to form new glycosidic bonds between short oligosaccharide units, thus contributing to the production of the LPS. As the LPS is highly specific for each bacterial species and strain, inhibitors of its biosynthetic route would be selective against a specific bacterium. Understanding Wzy structure would be a step forward in the rational design of novel antibiotics. In this study, Wzy was cloned and expressed in E. coli as a chimera with GFP and His tags. The protein was solubilized in different detergents and purified with chromatographic methods. The purification results allowed to identify the optimal detergents to obtain a sample suitable for crystallization or EM analysis. Stability of the protein was tested against a temperature gradient with Circular Dichroism, Raman spectroscopy, and fluorescence spectroscopy. With CD and fluorescence spectroscopy, a melting point of about 58\ub0 C was determined. From crystallization trials different crystals were obtained and analyzed at the XRD2 beamline of the Elettra Synchrotron, and data up to 6 \uc5 resolution were obtained. Structure solution revealed that crystals were formed by a contaminant present in the sample, the Cytochrome o Ubiquinol Oxidase. Further purification experiments suggested a tight interaction between Wzy and the Cytochrome o Ubiquinol Oxidase. Negative staining EM analysis was carried out on the protein-contaminant sample, confirming that the sample is monodisperse and homogeneous and is therefore suitable for further EM analysis

Azelaic Acid: A Bio-Based Building Block for Biodegradable Polymers

The production of fine chemicals, new materials and products from renewable feedstocks represents a continuous challenge. Several procedures have been reported in the literature or patented in the last decade for the main biomass components: carbohydrates (75%), lignins (20%), fats and oils (5%) [1]. Regarding oleochemical developments, the oxidative cleavage of unsaturated fatty acids to produce dicarboxylic acids, hydroxy acids, and amino acids has received great attention in the last decade [2]. Two main oleochemical products obtained by the cleavage of unsaturated fatty acids are sebacic acid and azelaic acid. Azelaic acid (AzA) is a naturally occurring saturated nine carbon atom dicarboxylic acid found in whole grains, wheat, rye and barley [2], first detected in rancid fats. It can be formed endogenously from substrates such as longer-chain dicarboxylic acids and processes like the metabolism of oleic acid, and ψ-oxidation of monocarboxylic acids. The azelaic acid market is predicted to reach USD 160 million by 2023 and the applications include pharmacological ingredients, polymers, plastics, lubricants and materials for electronics [3]. The aim of the present review is to highlight the potential of azelaic acid as powerful building block for the synthesis of bio-based and biodegradable polymers, with a special emphasis on the green synthetic routes, embracing both chemical and enzymatic methods

Turning biomass into functional composite materials: rice husk for fully renewable immobilized biocatalysts

Rice husk is an underexploited, low density and highly robust composite material, massively available from rice processing. Here we report two new procedures for the formulation of immobilized lipases applicable in fats and oils transformations. The enzymes were covalently anchored on aldehyde groups introduced on rice husk by laccase-catalysed oxidation of the cellulose component. The method avoids the use of toxic glutaraldehyde while allows for the application and recycling of the biocatalysts in aqueous media. The second method used a fluidized bed granulator for the coating of the particles of rice husk (200\u2013400 m) in the presence of water-soluble binders. The formulations are mechanically stable and suitable for applications in different hydrophobic media. Both methods allow for the recovery and reuse of the rice husk at the end of the life cycle of the biocatalysts

Microwave-Assisted Cyclization of Unprotected Dipeptides in Water to 2,5-Piperazinediones and Self-Assembly Study of Products\uad and Reagents

Dipeptides and their cyclized 2,5-piperazinedione (or diketopiperazine, DKP) derivatives are attractive building blocks for supra\uadmolecular hydrogels. The Phe-Phe, (p-nitro)-Phe-Phe, and Phe-Val dipeptides and their corresponding DKPs are studied for self-assembly in water. The DKPs were obtained in high yields by microwave-assisted cyclization\uad of the dipeptides in water, demonstrating that use of their methyl ester derivatives as reported in the literature is not necessary for successful cyclization. Single-crystal XRD structures are reported for two DKPs as well as stable hydrogels at neutral pH

Peptide stereochemistry effects from pKa-shift to gold nanoparticle templating in a supramolecular hydrogel.

The divergent supramolecular behavior of a series of tripeptide stereoisomers was elucidated through spectroscopic, microscopic, crystallographic and computational techniques. Only two epimers were able to effectively self-organize into amphipathic structures, leading to supramolecular hydrogels or crystals, respectively. Despite the similarity between the two peptides’ turn conformations, stereoconfiguration led to different abilities to engage in intramolecular hydrogen bonding. Self-assembly further shifted the pKa of the C-terminal side chain. As a result, across the pH range 4-6, only one epimer predominated sufficiently as a zwitterion to reach the critical molar fraction allowing gelation. By contrast, the differing pKa values and higher dipole moment of the other epimer favored crystallization. The four stereoisomers were further tested for gold nanoparticle (Au NP) formation, with the supramolecular hydrogel being key to control and stabilize Au NPs, yielding a nanocomposite that catalyzed the photodegradation of a dye. Importantly, the Au NP formation occurred without the use of reductants other than the peptide, and the redox chemistry was investigated by LC-MS and NMR. This study provides important insights for the rational design of simple peptides as minimalistic and green building blocks for functional nanocomposites