The Effects of Zinc Oxide Nanoparticle Interface on Conformational Dynamics of Protein Models From Different Structural Hierarchies

In recent years, nanoparticles and nanomaterials have found their far reaching applications in various fields including the field of biology and medicine. The consequences of these nanoparticles in biological milieu need to be properly assessed. As soon as a nanoparticle enters a biological milieu, a myriad of changes take place. Proteins present in milieu get adsorbed and desorbed over the nanoparticle interface in a dynamic process, resulting in a protein corona. Despite the advances made in nanosciences, our understanding of interactions between protein and nanoparticle interface is still limited. Nanoparticle interface behaviour is anticipated to change with change in accessible surface behaviour of the proteins present in biological milieu. Hence, it becomes essential to study the impact of nanoparticle interface on conformational and amyloidogenic properties of proteins with varying surfaces present in biological milieu. Thus, the thesis discusses the observed effects of zinc oxide nanoparticle (ZnONP) interface on conformational and amyloidogenic propensities of protein models belonging to structurally different hierarchies. Initially, the thesis shows changes in conformational and amyloidogenic propensities for an intrinsically disordered polypeptide (IDP), like IAPP, with change in the length of negatively charged polymeric surfaces, i.e. heparin fragments; diffusive binding of smaller heparin fragments through IAPP sequence is anticipated to delay the fibrillation, whereas interactions with longer fragments (> heparin heptamer) stabilize N- and C- terminus charged residues and expose IAPP self-recognition element resulting in enhanced fibrillation. On the other hand, ZnONP with negative surface potential interaction with monomeric IAPP found to inhibit the fibrillation and the fibril-mediated cytotoxicity. The second part of the thesis indicates the effect of ZnONP interface on another, relatively longer, IDP, i.e. -synuclein. The results indicated that highly favorable interaction between the interface and protein forms thermodynamically stable complex resulting into amorphous aggregation, instead of fibrillation; the interaction must have raised the threshold barrier between the structures, complex and amyloid structures, resulting the complex to kinetically trap in amorphous aggregate. However, the interaction with globular protein like insulin showed opposite results, which is discussed in subsequent chapter of the thesis. The interaction of insulin with ZnONP interface results in protein conformational rearrangement into an amyloid-prone conformation. The conformation fibrillates relatively faster and causes enhanced fibril-mediated cell death on increasing the interface concentration in solution at physiological pH. Otherwise, the protein at higher concentrations only forms amorphous aggregate in physiological pH. The thesis ends with the discussion on the effects of interface interaction with quaternary protein, like Concanavalin A (ConA). Interestingly, ConA adopts different unfolding conformations upon interaction with different chaotropes, like SDS, guanidinium chloride etc. Guanidinium chloride completely unfolds the protein above 2 M, whereas sodium dodecyl sulfate took protein into all-α protein from an all- protein. Additionally, ConA interaction with ZnONP interface causes conformational rearrangement with relatively more exposed hydrophobic patches, resulting into amorphous aggregation of the protein. Thus, the thesis findings, altogether, indicate that the interacting interfaces, like ZnONP with negative interfacial potential and protein interface, predominantly determine conformational changes in protein upon interaction, and its subsequent consequences like amyloidosis, flocculation

Conformation and aggregation of selectively PEGylated and lipidated gastric peptide hormone human PYY3–36

The gastric peptide hormone human PYY3–36 is a target for the development of therapeutics, especially for treatment of obesity. The conformation and aggregation behavior of PEGylated and lipidated derivatives of this peptide are examined using a combination of fluorescence dye assays, circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC) measurements, small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM). The behavior of two PYY3–36 derivatives lipidated (with octyl chains) in different positions is compared to that of two derivatives with PEG attached at different residues and to that of the native peptide. We find that, unexpectedly, PYY3–36 forms amyloid fibril structures above a critical aggregation concentration. Formation of these structures is suppressed by PEGylation or lipidation. PEGylation significantly reduces the (reversible) loss of α-helix content observed on heating PYY3–36. The PEG conjugates form mainly monomeric structures in solution- coiled-coil formation, and other aggregation presumably being sterically hindered by swollen PEG chains. However, some small aggregates are detected by AUC. In complete contrast, both of the two lipidated peptides show the formation of spherical micelle-like structures which are small oligomeric aggregates. Our findings show that PEGylation and lipidation are complementary strategies to tune the conformation and aggregation of the important gastric peptide hormone human PYY3–36

Self-Assembly of Synthetic and Biological Polymeric Systems of Technological Interest

In the present work, we investigated the micellization, gelation and the structure of aggregates of three poly(ethylene oxide)-poly(styrene oxide)-poly(ethylene oxide) block copolymers (EO12SO10, EO10SO10E10 and EO137SO18EO137, where E represents the oxyethylene unit, S the oxystyrene unit, and the subscripts correspond to the number of monomeric unit constituting the polymeric chain) in solution. We also investigated the adsorption process and the surface properties of these block copolymers at the air-water (a/w) and chloroform-water (c/w) interfaces. Since these copolymers possess a more hydrophobic middle block –the styrene oxide unit, they should gives rise to more efficient drug delivery systems, in which geometry might play a key role for drug solubilisation and cell uptake. For these reasons, a detailed characterization of the physicochemical properties of these copolymers is required

Towards the elucidation of pathophysiology of amyloid conversion of globular proteins

Amyloidoses are a group of diseases caused by the conversion of soluble proteins into pathogenic ordered fibrillar aggregates. The mechanism driving in vivo the structural transformation of these proteins has not yet been clearly elucidated. My work has focused on two plasma proteins that make amyloid in vivo starting from precursors deeply different in terms of structure and function: transthyretin (TTR) and the apolipoprotein C-III variant, D25V (D25V apo C-III). Apo C-III is mostly synthesized by the liver and is a major component of HDL. We have described the first variant causing a genetic form of renal amyloidosis. In the work presented here, structural and functional characterization of the newly described D25V apo C-III variant was carried out together with the investigation of its aggregation mechanism, showing a modest loss of function and an increased tendency to aggregate in physiological buffer in the lipid-free state. TTR is mainly synthesized by the liver and the choroid plexus in the CSF and is the main transporter of thyroxine in the CSF and the secondary transporter in plasma. The mechanism of TTR fibrillogenesis has been investigated for decades. Our group has recently proposed a new pathway for TTR amyloidogenis mediated by selective tryptic cleavage, in alternative to the commonly accepted low pH induced aggregation mechanism. Further characterization of the mechanism, including the identification of the culprit protease responsible for proteolytic cleavage in vivo was carried out showing a correlation between TTR stability and susceptibility to proteolysis. The inhibitory activity of stabilisers and their effect on protein structure and dynamics were also studied using a combination of spectroscopic techniques including NMR. The identification of the enzyme responsible for cleavage in vivo, opens up a completely new scenario for understanding the mechanism and the history of the disease in vivo

Supramolecular Systems for Gene and Drug Delivery

Dear Colleagues,Supramolecular systems (calixarenes, cyclodextrins, polymers, peptides, etc.) have attracted special attention due to their excellent therapeutic properties for biomedical applications such as gene and drug delivery. Numerous biomaterials-based supramolecular systems have been developed in the last decade for enhancing of biocompatibility and pharmacological activity. In particular, supramolecular nanomaterials are considered a hot research topic, because nanomedicine has become an interesting tool for the treatment of genetic diseases or cancer. Nevertheless, novel systems and their properties are being continuously studied, contributing to the development of efficient delivery systems.This Special Issue provides and highlights current progress in the use of the supramolecular systems for boosting gene and drug delivery. Preparation, characterization, and use of these systems, as well as the latest developments in this research field, are especially welcome.Authors are encorauged to submit original research articles and reviews in this promising research field

Rational design of amphiphilic fluorinated peptides: evaluation of self-assembly properties and hydrogel formation

Advanced peptide-based nanomaterials composed of self-assembling peptides (SAPs) are of emerging interest in pharmaceutical and biomedical applications. The introduction of fluorine into peptides, in fact, offers unique opportunities to tune their biophysical properties and intermolecular interactions. In particular, the degree of fluorination plays a crucial role in peptide engineering as it can be used to control the characteristics of fluorine-specific interactions and, thus, peptide conformation and self-assembly. Here, we designed and explored a series of amphipathic peptides by incorporating the fluorinated amino acids (2S)-4-monofluoroethylglycine (MfeGly), (2S)-4,4-difluoroethylglycine (DfeGly) and (2S)-4,4,4-trifluoroethylglycine (TfeGly) as hydrophobic components. This approach enabled studying the impact of fluorination on secondary structure formation and peptide self-assembly on a systematic basis. We show that the interplay between polarity and hydrophobicity, both induced differentially by varying degrees of side chain fluorination, does affect peptide folding significantly. A greater degree of fluorination promotes peptide fibrillation and subsequent formation of physical hydrogels in physiological conditions. Molecular simulations revealed the key role played by electrostatically driven intra-chain and inter-chain contact pairs that are modulated by side chain fluorination and give insights into the different self-organization behaviour of selected peptides. Our study provides a systematic report about the distinct features of fluorinated oligomeric peptides with potential applications as peptide-based biomaterials

The Effect of RNA Nucleic Acid Aptamers on the Aggregation of Green Fluorescent Protein

Protein aggregation can impact the product quality of pharmaceutical drugs in terms of efficacy, safety, and immunogenicity leading to economic and technical problems for biotechnology and pharmaceutical companies. Furthermore, protein aggregation can lead to the onset of a variety of diseases including amyloidosis, prion diseases, and other protein deposition disorders. To combat these issues, we investigated the potential of aptamers to prevent protein aggregation within our model, modified fluorescent protein. We have identified and successfully demonstrated that the previous selected AP3 aptamer has a high affinity for not only green fluorescent protein (GFP), but also to emerald GFP with a KD of 199 nM. Our work has successfully demonstrated that protein aggregation can be inhibited upon binding of RNA aptamers

Formation and properties of whey protein fibrils

Protein fibrils are threadlike aggregates that are about one molecule thick and more than thousand molecules long. Due to their threadlike structure they could potentially be used to form meat-like structures. Protein fibrils can be produced from milk protein and plant protein, opening opportunities for a more sustainable food production. For a successful application of the fibrils it is important to know how fibrils are formed and how to influence the properties of the fibrils. This thesis describes how fast fibrils are formed and determines the energy change involved in this formation. Fibrillar structures show promise as encapsulating material, thickener, gelling and flocculation agent. This thesis provides new insights that facilitate innovations in the area of tasty, healthy and sustainably produced food. </p