Lipase activity in vesicular systems: Characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation

DNA-Based Mimics of Membrane Proteins Lipid-DNA Interactions Determine Function

Nucleic acids, particularly DNA, are used as a nanoscale building material, due to their unique controllability via complementarity of base pairing. One of the potential applications of DNA nanotechnology is creating synthetic constructs mimicking function of membrane proteins. These natural molecular machines function embedded in the lipid bilayer. Similar membrane attachment of DNA-based structures is achieved by modifying the nucleic acid with hydrophobic anchors, most commonly cholesterol. Aiming at developing a fully functional and controllable synthetic membrane construct, the first step I undertook was to understand and utilize fundamental interactions between molecules: DNA, cholesterol and lipids. Instead of starting with a complicated DNA-based model mimicking protein architecture, here I have created a set of simple systems that allowed me to examine the major interactions between involved molecules. This work describes four aspects of the DNA-lipid systems that I have built and studied experimentally. Firstly, I have analysed the effects of membrane-spanning DNA duplex on the lipids’ arrangement in the pore and presented how this arrangement can be remodelled depending on the hydrophilicity of the DNA design. Secondly, I have looked at the same system from the opposite perspective - studied and prevented the distortion of the transmembrane DNA construct induced by the surrounding lipids. Thirdly, I have evaluated the importance of ions in mediating DNA-lipid interactions, reporting analysis of two electrostatic phenomena: screening and bridging. Finally, utilizing a nanoengineered four-helix structure, I discussed surfactant’s influence on DNA membrane insertion efficiency, showing that aggregation of the nanostructures is one of the major factors determining their spontaneous membrane-spanning. While the understanding of phenomena in minimalistic systems is crucial for further development of complex pore-forming constructs, here I showed that even simple DNA nanostructures, when rationally designed, can mimic functionality of natural membrane proteins.EPSRC; Winton Programme for the Physics of Sustainabilit

Efectos de las proteínas SP-B y SP-C del surfactante pulmonar en las propiedades físicas de membranas biológicas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Físicas, Departamento de Física Aplicada III, leída el 10/07/2013Depto. de Estructura de la Materia, Física Térmica y ElectrónicaFac. de Ciencias FísicasTRUEunpu

EFFECT OF HYDRATION AND MACROMOLECULAR CROWDING ON PEPTIDE CONFORMATION, AGGREGATION AND FOLDING KINETICS

Protein folding/misfolding in vivo takes place in a highly crowded and confined environment. Such crowded environment can possibly lead to fewer water molecules surrounding a protein of interest than that seen under in vitro conditions wherein typically dilute aqueous solutions are used. When considering the aforesaid cellular characteristics, such as water depletion and macromolecular crowding; it is reasonable to assume that proteins may experience different energy landscapes when folding in vivo than in vitro. Therefore, we have investigated how degrees of hydration and macromolecular crowding affect the conformation, aggregation and folding kinetics of short peptides. In order to modulate the number of water molecules accessible to the peptide molecules of interest, we encapsulated the peptides in the aqueous core of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and isooctane (IO) at different water loadings. Using this reverse micellar platform, we systematically studied the conformation and aggregation properties of alanine-based peptides and amyloid forming segments derived from amyloid beta peptides and yeast prion protein Sup35 at different hydration levels. Our studies demonstrated that limited hydration facilitates aggregate formation in these peptides and that removal of water imposes a free energy barrier to peptide association and aggregation. These studies have implications for understanding aggregate/amyloid formation in vivo where macromolecular crowding can change the solvation status of the peptides. Furthermore, we examined how the folding dynamics of secondary/supersecondary structural elements are modulated by a crowded environment in comparison to that of dilute aqueous solutions. To this effect we studied the thermal stability and folding-unfolding kinetics of three small folding motifs, i.e., a 34-residue alpha-helix, a 34-residue cross-linked helix-turn-helix, and a 16-residue beta-hairpin, in the presence of crowding agents (i.e. inert high mass polymers). Our results indicate that the folding-unfolding transition of alpha-helical peptides is insensitive to macromolecular crowding. However, we find that crowding leads to an appreciable decrease in the folding rate of the shortest beta-hairpin peptide. We propose a model considering both the static and dynamic effects arising from the presence of the crowding agent to rationalize these results

Recent advances in smart biotechnology: Hydrogels and nanocarriers for tailored bioactive molecules depot

Over the past ten years, the global biopharmaceutical market has remarkably grown, with ten over the top twenty worldwide high performance medical treatment sales being biologics. Thus, biotech R&D (research and development) sector is becoming a key leading branch, with expanding revenues. Biotechnology offers considerable advantages compared to traditional therapeutic approaches, such as reducing side effects, specific treatments, higher patient compliance and therefore more effective treatments leading to lower healthcare costs. Within this sector, smart nanotechnology and colloidal self-assembling systems represent pivotal tools able to modulate the delivery of therapeutics. A comprehensive understanding of the processes involved in the self assembly of the colloidal structures discussed therein is essential for the development of relevant biomedical applications. In this review we report the most promising and best performing platforms for specific classes of bioactive molecules and related target, spanning from siRNAs, gene/plasmids, proteins/growth factors, small synthetic therapeutics and bioimaging probes.Istituto Italiano di Tecnologia (IIT)COST Action [CA 15107]People Program (Marie Curie Actions) of the European Union's Seventh Framework Program under REA [606713 BIBAFOODS]Portuguese Foundation for Science and Technology (FCT) [PTDC/AGR-TEC/4814/2014, IF/01005/2014]Fundacao para a Ciencia e Tecnologia [SFRH/BPD/99982/2014]Danish National Research Foundation [DNRF 122]Villum Foundation [9301]Italian Ministry of Instruction, University and Research (MIUR), PRIN [20109PLMH2]"Fondazione Beneficentia Stiftung" VaduzFondo di Ateneo FRAFRAinfo:eu-repo/semantics/publishedVersio

A Langmuir approach on monolayer interactions to investigate surface active peptides

The Langmuir Blodgett apparatus provides a versatile system for studying the interfacial properties of peptides and peptide-membrane interactions under controlled conditions. Using amphiphilic α-helical peptides to highlight studies undertaken, here we discuss the use of this system to provide information on the surface activity of peptides and describe the insights these studies give into biological functio

New Methods To Assess Protein Folding And Conformational Dynamics

A protein’s folding and conformational energy landscape depends on a large number of molecular degrees of freedom and interactions. As a result, different proteins can follow different sequences of events moving toward the native state along the course of folding. For example, the underlying structural organization and ordering can occur locally first and then globally, or vice versa. In addition, the associated conformational transitions can take place over a wide range of timescales. Because of these complexities, arriving at a detailed assessment and understanding of the folding dynamics and mechanism of any protein via a single type of experiment is challenging, and sometimes impossible. As such, over the past two decades, many different experimental methods have been employed to study how proteins fold among which, the laser-induced temperature-jump (T-jump) technique has emerged as a powerful tool to measure protein folding kinetics occurring on the nanosecond and microsecond timescales. Herein, we further expand the utility of the T-jump technique. First, we introduce a new form of the T-jump technique (referred to as VIPT-jump) that can be used to distinguish between different folding mechanisms. Second, we apply the VIPT-jump concept to better understand the folding dynamics of an alanine-based -helix, and, in conjunction with theoretical modeling, we are able to determine the long-sought microscopic rate constants of the helical nucleation and propagation processes. Third, we develop a new method to extend the time window of observation in a T-jump experiment to the millisecond timescale. In a parallel effort, we demonstrate that quenching the fluorescence of a dye molecule by a tryptophan residue via photoinduced electron transfer mechanism can be used to interrogate the conformational dynamics of proteins that are crucial for function. Applying this method to the M2 proton channel of the Influenza A virus allow us to determine, for the first time, the gating dynamics of the tryptophan tetrad in this membrane protein

Protein separation using surfactant precipitation

Surfactant precipitation applied as a surfactant mediated protein purification technique has considerable potential in protein extraction, and therefore the understanding of the interactions involved and the folding behaviour in the precipitated protein was the first aim of this thesis. The key system parameters such as buffer salt concentration, molar ratio of surfactant to protein and pH which determines the protein stability in protein-surfactant complex formation were evaluated. The surfactant:protein ratio determines saturation of protein binding sites while pH determines the strength of affinity for ionic binding which influences hydrophobic binding with surfactant monomers causing the protein to lose its conformation. The protein-surfactant binding varied for lysozyme, cytochrome c and ribonuclease A with trypsin and α -chymotrypsin, and hence the denaturation profile. In the second aim, protein recovery from surfactant precipitation was enhanced by improving the solvent recovery method and, implementing a new and novel counterionic surfactant recovery method. The effect of a variety of recovery phases and solution conditions on lysozyme recovery was analysed in terms of their ability in maintaining protein stability, recovery yield, and activity. It was found that solvent recovery was limited by solvent polarity and protein solubility, and that the cationic surfactant, trioctylmethylammonium chloride (TOMAC), used to form nonpolar ion pairs with sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) was the most efficient method for recovering protein. The third aim was to assess the influence of protein properties, such as charge and hydrophobicity, on protein separation. The selective extraction of a target protein from mixtures of proteins in both buffer and fermentation broth was investigated. It appears that the optimum surfactant:protein molar ratio for the extraction of the proteins from fermentation broth (lysozyme, cytochrome c and ribonuclease A; 16, 17 and 22 respectively) were similar to those in a buffer system. Lysozyme and ribonuclease A were selectively separated from a binary mixture. The extraction behaviour was well represented by surface charge distribution which is indifferent to system conditions. However, certain broth constituents induced the formation of some unfolded irreversible non-dissolvable precipitate in the recovery process. Finally, the use of non-ionic surfactants, ionic/non-ionic mixed surfactants, and cationic surfactants were investigated in surfactant precipitation system. Non-ionic surfactant does not support direct precipitation of proteins using surfactant or recovery of protein from a protein-surfactant complex, and has no effect in a mixed ionic/non-ionic system. The application of cationic surfactant precipitation to separate trypsin inhibitor was attempted, and good recovery was obtained

Electrostatic Effects on Lipid Bilayer Physicochemal Properties and Vesicle Adhesion

Lipids are an integral part of cells, being the principal component of the cell membrane, and contributing to the function and regulation of biological processes. Lipid nanoparticles mimicking a cell’s endosomes or exosomes are of particular interest within the pharmaceutical industry for their ability to deliver cargo such as RNA into target cells. The delivery process faces a multitude of challenges, so a rational design approach for vesicles that considers a lipid’s physicochemical contribution to the membrane is desired. To that end, this thesis explores the creation of large area biomembranes along with the development of electromechanical and optical characterization methods with the goal of determining structure-function relationships of membranes and their constituent lipids in different compositional and ionic environments. We demonstrated the ability to create large area model biomembranes (LAMBs) with control over their lipid and external ionic composition, both symmetrical and asymmetrical. We explored the Young’s modulus and bending rigidity of DOPC, DOPG symmetric and asymmetric membranes using electrostriction. This enables us to map the lipid-dependent physical property changes for bilayers, relate lipid composition to physical rearrangement such as formation of a fusion stalk, and compare them to existing literature for other platforms. The fusion process necessitates interaction between two opposing lipid monolayers; thus we use a thin film balance approach to explore headgroup-headgroup interactions. These interactions are modulated by electrostatic forces arising from two charged monolayers nearing each other, the ionic contents of the solution, and structural forces. We determine the magnitude of the electrostatic component of the film’s disjoining pressure through careful drainage. Additionally, dynamic drainage of the same films yields aggregate information of both electrostatic and structural forces within the film. Knowledge of monolayer headgroup interactions will help inform and shape the design space for vesicle-membrane fusion experiments Towards this end, we have performed experiments exploring calcium induced vesicle growth, adhesion of vesicles on the bilayer surface, and demonstrated analytical approaches to quantify deposition, growth, and fusion. Cationic lipid within the membrane promotes adhesion and retention of vesicles. The work laid out here establishes the design of vesicle-bilayer systems that can be expanded to more complex biologically relevant compositions