INVESTIGATING PROTEIN - BILAYER COMPLEXES: A STUDY OF LIGAND - RECEPTOR INTERACTIONS AT MODEL MEMBRANE SURFACE BY USING ELECTRONIC ABSORPTION SPECTROSCOPY AND FLUORESCENCE RESONANCE ENERGY TRANSFER.

Author

Navneet Dogra, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Kohli, Punit

Abstract

The main aim of work presented here is to design, develop and characterize a colorimetric model membrane (liposome) systems, which can bind with proteins, enzymes, bacteria, virus and other biomolecules. PDA molecules are utilized as a scaffold for the bilayer membrane, and a colorimetric assay is carried out. The holy grail of present work contributes towards the better understanding of protein interactions with the cell bilayer surface. Chapter 1 introduces a brief history on the advent of bilayer systems for cellular research exploration. We presented a literature survey about how liposome systems are used as a complementary technique to understand the fundamental principles of cellular membrane functions. Furthermore, we describe about membrane protein functions and recent findings on how proteins interact with the cell membrane. Finally, we explain conjugated systems and their exploration in bilayer membrane as a colorimetric scaffold. We also touch bases with major fluorescence techniques used in our experiments. Chapter 2 provides details on the preparation protocols of liposome and liposome-protein complexes. We confirmed protein-bilayer interactions by monitoring FRET between PDA and rhodamine molecules. Furthermore, we performed streptavidin-biotin binding studies on the PDA bilayer. Protein binding changed the spectral overlap (J) between PDA and rhodamine, which ultimately increased the fluorescence emission of rhodamine. The goal of performing these studies was to present a complete protocol for the preparation of liposome and protein-liposome complex. In chapter 3, we investigate how proteins bind on the cell membrane. Additionally, we propose a model of protein-bilayer complex. We reported that, by harnessing cell bilayer with specific bio-molecules, we monitored protein--bilayer, protein--protein and enzyme--substrate signal transduction. We have developed a colorimetric system for monitoring vital stimulations occur on the liposomal membrane surface. Bilayer was modified to covalently bind the amino group of lysine residues present on protein molecules. These bio-molecular interactions on bilayer surface provide differential stimulus, which turned out to be the major cause of differential spectroscopic signals depending upon size and shape of the protein bounded to the bilayer. Polydiacetylene (PDA) liposomes are the core of our color based system. These liposomes are used to monitor subtle interactions on the bilayer surface. We have also developed a semi-quantitative method based on the colorimetric response of PDA liposomes; we were able to detect protein molecules at sub-nanomolar concentrations in the solution. It's capability of distinguishing protein molecules based on their chemical and physical interactions to bilayer contributes towards the identity of our system. Interestingly, our mass spectroscopic data suggested non-specific enzymatic cleavage of membrane-bound proteins. These fragments were not present in bulk protein cleavage. We also proposed a model that depicts the covalent binding of protein at the bilayer of liposomes. These studies are intended to investigate protein-bilayer and enzyme-protein interaction occurring on the cell surface. In chapter 4, we focus on the kinetics of protein interaction on bilayer surface and we also attempt to visualize these interactions by exploring fluorescence microscopy. A self-assembled cell membrane is consisted of various lipids, which cluster themselves in their preferred phase separated regions. Lipid clusters are very important for lipid specific protein interactions. We investigated protein binding on such phase separated regions under a fluorescence microscope. Furthermore, we enzymatically catalyzed proteins, which were covalently bonded on the bilayer surface. This catalytic reaction was monitored both spectroscopically and under a fluorescence microscope. These studies were performed to help us in the better understanding of biological interactions at cell surface. Chapter 5, describes the encapsulation and controlled delivery of antimicrobial compounds from liposomes. Use of antimicrobial coatings on food packaging is one of the important technologies of active packaging for improving food safety. There is growing demand for natural antimicrobials because of fear of adverse health effects of synthetic preservatives. The main objective of this study is to compare antimicrobial activity of free versus encapsulated curcumin. Glass surfaces coated with nano-encapsulated curcumin may be used as an active packaging material in preserving liquid foods; however, further study is required to improve antimicrobial activities of polylactic acid PLA surfaces. In chapter 6, we investigate interactions between receptors and ligands at bilayer surface of polydiacetylene (PDA) liposomal nanoparticles using changes in electronic absorption spectroscopy and fluorescence resonance energy transfer (FRET). We study the effect of mode of linkage (covalent versus noncovalent) between the receptor and liposome bilayer. We also examine the effect of size-dependent interactions between liposome and analyte through electronic absorption and FRET responses. Glucose (receptor) molecules were either covalently or noncovalently attached at the bilayer of nanoparticles, and they provided selectivity for molecular interactions between glucose and glycoprotein ligands of E. coli. These interactions induced stress on conjugated PDA chain which resulted in changes (blue to red) in the absorption spectrum of PDA. The changes in electronic absorbance also led to changes in FRET efficiency between conjugated PDA chains (acceptor) and fluorophores (Sulphorhodamine-101) (donor) attached to the bilayer surface. Interestingly, we did not find significant differences in UV−Vis and FRET responses for covalently and noncovalently bound glucose to liposomes following their interactions with E. coli. We attributed these results to close proximity of glucose receptor molecules to the liposome bilayer surface such that induced stress were similar in both the cases. We also found that PDA emission from direct excitation mechanism was ∼2−10 times larger than that of the FRET-based response. These differences in emission signals were attributed to three major reasons: nonspecific interactions between E. coli and liposomes, size differences between analyte and liposomes, and a much higher PDA concentration with respect to sulforhodamine (SR-101). We have proposed a model to explain our experimental observations. Our fundamental studies reported here will help in enhancing our knowledge regarding interactions involved between soft particles at molecular levels. In chapter 7, we conclude the summary of all work carried out in previous chapters.