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Author Kamilya, Tapanendu
Source IACS Kolkata
Content type Text
Educational Degree Doctor of Philosophy (Ph.D.)
File Format PDF
Language English
Subject Domain (in DDC) Natural sciences & mathematics
Abstract Interactions of proteins with lipids are the area of fundamental interest due to enormous biological importance. From the biotechnological and biomedical applications point of view, studies on lipids and proteins have been playing a significant role in developing protocols for drug delivery and immunosensing system, various biomolecular devices etc. The immobilization of protein without denaturation on solid support, or on lipid membrane, is extremely valuable and is the fundamental step for designing such devices.This dissertation entitled as “STUDIES OF PROTEIN LIPID INTERACTION AT AIR/WATER INTERFACE BY LANGMUIR-BLODGETT TECHNIQUE” deals with some studies on lipids, proteins/enzymes with the aim to understand various aspects of lipid-protein interaction. The main thrust of the work involves the development and characterization of protein/protein-lipid films on air/water interface and subsequent transfer on a solid substrate. Besides the characterization, the aim is also to minimize the aggregation of protein in lipid matrix as well as to prepare the protein-lipid mixed film with networks of individual protein molecules. Langmuir-Blodgett (LB) and Self Assembly (SA) techniques have been used to prepare the desired systems. Moreover, combination of different techniques are used to comprehend the basic science behind the lipid/protein film formation, influence of surface treatment and protein/enzyme interaction, the role of net charge of the protein/lipid etc. This dissertation comprises of seven chapters and one appendix. Chapter 1 elucidates the necessities for studying the scientific interdisciplinary works. It highlights the need of studies on the adsorption of protein at bare air/water interface and into lipid membrane. It focuses on the interaction between lipids and proteins and the necessities for immobilization of proteins and protein-lipid complexes onto solid supports with minimum aggregation and denaturation of protein. It also contains a general introduction with a brief overview of relevant parts of existing works and concepts required to interpret the experimental results.Chapter 2 focuses on the experimental techniques, which are used for the formation of enzyme/protein, and protein-lipid mixed thin film. It also describes the different characterization techniques to study the enzyme/protein and protein-lipid mixed film.Chapter 3 focuses on the effect of salt (KCl) on the interfacial surface activity and the formation of yeast alcohol dehydogenease (ADH) monolayer at air/water interface using Langmuir-Blodgett technique. It elucidates the effect of salt on the formation of ADH monolayer. It also highlights on the compressibility, rigidity and unfolding of ADH at air/KCl-water interface. The observed significant changes above 0.1M KCl concentrations are explained in context of the DLVO theory. CD and FTIR spectroscopic studies together with AFM imaging of ADH monolayer indicate that KCl perturbs the ADH monolayer by incrementing the -structure resulting into larger unfolding and intermolecular aggregates at high salt concentration.Chapter 4 elucidates the surface activity of an enzyme, pepsin at air/water interface with salt (KCl), pH variation in subphase as well as the incorporation into a cationic octadecylamine monolayer. It also highlights on the area/molecule, compressibility, rigidity, unfolding of pepsin at air/KCl-water interface with pH closest to its isoelectric point (optimized subphase) as well as into the lipid monolayer. The structural aspects of deposited pepsin films at different conditions are analyzed by using FE-SEM and FTIR spectroscopy to study the conformational change of Pepsin, due to thin film formation by LB technique at different environmental conditions.Chapter 5 describes the comparative study of the incorporation of a protein; ovalbumin (OVA) within octadecylamine (ODA), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and stearic acid (STA) suggests that it is more preferable in case of ODA (cationic) than DPPC (zwitterionic) and STA (anionic) lipid monolayer due to electrostatic interaction. The incorporation of OVA into different monolayers is found to be surface pressure dependent and more in LE region than in condensed region. Addition of STA and ODA quenches tryptophan fluorescence by perturbing tyrosine excitation. However, addition of DPPC does not perturb tyrosine moieties, indicates a little perturbation of OVA structure. Tyrosine may be the binding site with STA as well as ODA. Although higher accumulation of OVA occurs in case of ODA monolayer yet FE-SEM and fluorescence study supports that DPPC monolayer is better to get protein lipid mixed film than STA and ODA monolayer. It also suggests that the extent of aggregation of protein in protein-lipid mixed film is less in zwitterionic lipid monolayer however can not be minimized by LB film deposition technique. Chapter 6 deals with a comparative study on the films fabricated by self-assembly (SA) of OVA-DPPC vesicle on glass substrate and SA of OVA on prefabricated LB film of DPPC. It also focuses on the immobilization of DPPC and OVA-DPPC vesicles on solid substrate by simple drop-cast technique. FTIR analyses of amide bands are being used to inspect the extent of the conformational transitions of -sheet to -helix of OVA in immobilized DPPC vesicles. FE-SEM imaging of immobilized OVA-DPPC vesicles provide the structure of individual OVA molecule attached with DPPC, without aggregation amongst them.Chapter 7 describes the concluding remarks of overall study and future perspectives. Appendix contains a list of publications and presentations.
Education Level UG and PG
Learning Resource Type Thesis