Vibrational Spectroscopic studies of Dynamics of BSA unfolding, Lysozyme-Ethanol interaction and Tissue Imaging
Vibrational spectroscopy has served as a powerful tool for understanding various structural properties of the molecules since decades. In the recent years, it has also emerged as an invaluable tool in understanding various biological processes as well. Apart from understanding various fundamental processes like protein folding, DNA-drug interaction, protein-protein interaction, protein unfolding etc at the molecular level, it has found ample scope in understanding higher biological entities like cells, tissues body fluids etc. These techniques are rapid, non-destructive and offer multiple component analysis (global/multiplex) in a single measurement without any labels and can be an efficient and effective tool available to scientists. The subject of this thesis is the demonstration of the applicability of the various vibrational spectroscopic techniques like Raman spectroscopy, Infrared spectroscopy and Raman Optical activity to understand various biophysical and biochemical properties concerning various biomolecules and more complex bio-systems like tissues. The present thesis has been divided into six chapters and primarily deals with application of various vibrational spectroscopic techniques (Raman, Infrared, and Raman Optical Activity) to Bovine Serum Albumin (BSA), Lysozyme and oral cancer tissues. Chapter 1 gives brief literature review of the applications of various vibrational spectroscopic techniques in the field of biology (biomolecules and higher bio-assemblies). Biomolecules are essential building blocks of life. They are central to all life processes and are involved in carrying out important metabolic reactions and maintaining the overall biochemistry of a living organism. Different kinds of biomolecules like nucleic acids, proteins, lipids and carbohydrates are involved in maintaining the functional homeostasis of a cell. Interestingly, each of these biomolecules has distinct chemical identities and therefore has unique molecular structures. Consequently, these biomolecules generate a signature Raman spectrum. Any biological material (cell, tissue etc) is made up of different composition of various biomolecules. A prior knowledge about the biomolecules can help in understanding the composition of different cells, tissues etc. Different cells, tissues, bio-fluids also have unique vibrational spectra which can help in understanding the changes occurring due to any disease or any other pathological state. Chapter 2 deals with the principle of vibrational spectroscopy, instrumentation and various experimental difficulties encountered during vibrational spectroscopic measurements. Although Raman spectra of molecules can be quite informative, interpretation of these spectra can be complicated due to various overlapped bands. Therefore, various data analysis techniques are used. Two-dimensional correlation analysis is one such important data analysis method which not only helps in resolving the various overlapped bands but also gives useful information about the in-phase and out-of-phase correlation of various bands. Chapter 3 and 4 present a model study to understand the denaturation and ligand interactions of proteins. Proteins are one of the most essential building blocks of life and have numerous functions in body. It is the three-dimensional architecture of proteins which makes it so specific and selective for a biological function. If a protein fails to reach its native state it can no longer fulfil its biological function. Protein misfolding brings plenty of unknown facts to be understood like why are misfolded proteins toxic, what are their structural features and cellular targets, which properties of proteins are disease-specific and how does cellular mechanism fights or fails to neutralize such proteins? A large number of biophysical techniques are used for studying protein folding and protein-ligand interaction like Nuclear Magnetic Resonance spectroscopy, crystallography, circular dichroism, fluorescence etc. From the past few years, vibrational spectroscopy has also been used to study proteins. Vibrational spectroscopy has been widely used for structure elucidation of small molecules. While understanding the absolute structure of any protein can be a confounding task, monitoring changes in the chemical structure in response to some perturbation can be efficiently analysed using Raman spectra. Chapter 3 of the thesis presents Raman spectroscopic investigations of denaturation of Bovine Serum Albumin (BSA) using guanidinium hydrochloride (GuHCl). BSA is mostly alpha (α) helical in native state. Denaturation of BSA follows the melting of helices and appearance of more random structure of beta (β) sheets. Further, the data has been analysed using two-dimensional correlation analyses to understand relative in phase and out of phase correlation among various secondary structures like α helices, β sheets, turns etc. Chapter 4 focuses on the interaction of ethanol with Hen Egg White Lysozyme (Lysozyme) using Raman, Raman Optical Activity (ROA) and Molecular Dynamic Simulation studies. ROA is a special Raman technique which measures the difference in the Raman intensity of chiral molecules that interact with the right and left circularly polarized light or vice versa. ROA measures the signals that are often associated with vibrational coordinates that sample the most rigid and chiral parts of the structure and making spectra simpler. These are usually within the backbone and often give rise to ROA band patterns characteristic of the backbone conformation. In the first part of the chapter, Raman and ROA experiments have been performed which shows there in no denaturation up to 40% v/v of ethanol (amide I analysis). ROA studies suggest that there is an increase in the content of solvent-exposed helices. In the second part of chapter Molecular Dynamic Simulations (MDS) have been performed to understand the mechanistic of interaction of ethanol with lysozyme. Chapter 5 deals with FTIR imaging of oral cancer tissues of various grades. Every disease is associated with a change in biochemistry. This principle is the central doctrine for modern medicine. The biochemical changes can either be a cause for disease manifestation or may be a consequence of the disease itself. Since IR spectroscopy probes bond vibrations, the spectra generated are very molecule-specific. Any change in the concentration or the conformation of these biomolecules as a result of a disease, however subtle, is reflected in anIR spectrum. Progression of oral cancer has been analysed by studying the changes in IR spectra of oral cancer tissues of various grades. Prominent changes in various biomolecules like proteins, nucleic acids, glycogen can be observed during the progression of oral cancer. Finally, chapter 6 contains overall summary of the thesis and discusses future directions of the research work carried out in this thesis.