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Protein folding in crowded environments and living cells

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Title: Protein folding in crowded environments and living cells
Author(s): Dhar, Apratim
Director of Research: Gruebele, Martin
Doctoral Committee Chair(s): Gruebele, Martin
Doctoral Committee Member(s): McDonald, J. Douglas; Baranger, Anne M.; Prasanth, Kannanganattu V.
Department / Program: Chemistry
Discipline: Chemistry
Degree Granting Institution: University of Illinois at Urbana-Champaign
Degree: Ph.D.
Genre: Dissertation
Subject(s): protein folding live cells in vivo macromolecular crowding fast relaxation imaging Förster Resonance Energy Transfer (FRET) folding kinetics thermal denaturation
Abstract: Biomolecular dynamics and stability are predominantly investigated in vitro, and extrapolated to explain function in the living cell. In this thesis, we attempt to bridge this divide by performing studies of protein folding in in vitro crowded environments and in living cells. We begin by investigating the thermodynamic and kinetic behavior of the proteins in protein/carbohydrate matrices, and find significant differences compared to dilute, buffer solutions. We then develop Fast Relaxation Imaging (FReI) as a novel imaging technique to investigate protein folding dynamics inside living cells with millisecond temporal resolution and micrometer spatial resolution. We study the folding of the metabolic enzyme phosphoglycerate kinase (PGK) in U2OS (human osteosarcoma) cells and observe variations in folding kinetics within a single cell. FReI experiments carried out on an ensemble of 30 cells reveal large variations in folding time from cell to cell, which are analyzed to estimate the folding diffusion coefficient in vivo. Finally, we use short peptide tags to target PGK to the nucleus and endoplasmic reticulum of live cells and discern differential folding stability and kinetics in these organelles compared to protein folding in the cytoplasm.
Issue Date: 2011-05-25
Rights Information: Copyright 2011 Apratim Dhar
Date Available in IDEALS: 2011-05-25
Date Deposited: 2011-05

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