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Studies of protein folding and ligand binding dynamics in vitro and in cultured cells using fluorescence correlation spe correlation spectroscopy and allied biophysical techniques

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Title Studies of protein folding and ligand binding dynamics in vitro and in cultured cells using fluorescence correlation spe correlation spectroscopy and allied biophysical techniques
 
Creator Sarkar, Suparna
 
Subject Structural Biology & Bioinformatics
 
Description In order to function, newly synthesized protein need to fold
properly. Denatured proteins, which have had essentially all of their native three-dimensional structure disrupted, can refold from their random disordered state into a well-defined unique structure, in which the biological activity is virtually completely restored. During folding, a protein needs to search different possible conformations to reach into its native functional state. A comprehensive understanding of the early stages of protein folding remains elusive to date and is a subject of extensive research efforts. The major bottlenecks to study the early events of protein folding arise from the lack of computational and experimental techniques to study the unfolded and initial intermediate states. Fluorescence correlation spectroscopy (FCS) has been emerging as an important technique with single molecule resolution to study the diffusional and conformational dynamics of the early events of protein folding. In the present work, the rat intestinal fatty acid binding protein (IFABP) has been used as a model system. IFABP is a 15 kDa predominantly β-sheet protein that belongs to the intracellular lipid binding protein (iLBP) family. Fatty acid binding proteins are transport proteins delivering fatty acids (FAs) from cellular periphery to their subcellular destinations. They have been shown to play important roles in lipid metabolism and organelle dynamics. We used a combination of fluorescence correlation spectroscopy and far-UV circular dichroism (CD) to understand how the early processes like chain collapse and secondary structure formation influence the late folding events like the stabilization of the secondary structure and aggregation. Acid-induced unfolded IFABP was found to collapse in the presence of low concentrations of added salt and aggregate at higher concentrations. The results suggested that backbone hydrogen bond formation, not only the overall hydrophobicity of IFABP, may play crucial roles in the early collapse. Next, we explored the formation of intermediate state in the process of protein folding. We unfolded the protein by lowering the pH of the solution condition and monitored the occurrence of the intermediate state at around pH 3. A combination of tryptophan fluorescence and far-UV CD were used to study the unfolding of its tertiary and secondary structures. FCS was used to delve into the molecular events occurring in concert during the unfolding process. We observed that this intermediate state is stable enough to unfold its secondary structure after the unfolding of its tertiary structure in the presence of denaturing solvent like urea. We also observed that the effect of TFE upon the intermediate state is much less than the native or completely unfolded states. We carried over our in vitro work into the cellular system relating the structure-function properties of IFABP. A cellular model for lipid toxicity was generated with increased dose of a LCFA like sodium oleate. This lipid toxicity induced cell stress caused changes in mitochondrial morphology, loss in mitochondrial membrane potential and subsequent apoptosis. With controlled expression of IFABP in the HeLa cells, the mitochondrial morphology was partially restored with protection from apoptosis. The above findings were validated in Caco-2 cells where in the cellular metabolic stress, a controlled expression of IFABP could restore mitochondrial morphology and provided protection from apoptosis. A part of the thesis work also carries the distribution of IFABP in subcellular compartments in the presence and absence of its FA ligand and its diffusion was monitored at single cell resolution under cholesterol depleted condition by using CLSM, FCS, TIRF and FACS. It was noticed that although IFABP’s localization near the plasma membrane did not reduce under membrane cholesterol depleted condition, and the protein could still bind to some integral part of the membrane while receiving and trafficking its FA ligands inside the cell. Thus it was presumed that this protein could have evolved to receive their lipid ligands from the cell plasma membrane in a membrane microdomain-independent phenomenon. This present work is just a preliminary approach in addressing this phenomenon and more work are to be done to explore the mechanism of ligand uptake at single cell resolution.
 
Date 2014
 
Type Thesis
NonPeerReviewed
 
Format application/pdf
 
Identifier http://www.eprints.iicb.res.in/2091/1/Suparna_Sarkar_Ph.D._Thesis.pdf
Sarkar, Suparna (2014) Studies of protein folding and ligand binding dynamics in vitro and in cultured cells using fluorescence correlation spe correlation spectroscopy and allied biophysical techniques. PhD thesis, Calcutta University.
 
Relation http://www.eprints.iicb.res.in/2091/