University of Illinois Urbana-Champaign

Protein folding in crowded environments and living cells

Dhar, Apratim

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Dhar_Apratim.pdf

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https://hdl.handle.net/2142/24043

Description

Title
Protein folding in crowded environments and living cells
Author(s)
Dhar, Apratim
Issue Date
2011-05-25T14:58:42Z
Director of Research (if dissertation) or Advisor (if thesis)
Gruebele, Martin
Doctoral Committee Chair(s)
Martin Gruebele
Committee Member(s)
  • McDonald, J. Douglas
  • Baranger, Anne M.
  • Prasanth, Kannanganattu V.
Department of Study
Chemistry
Discipline
Chemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Date of Ingest
2011-05-25T14:58:42Z
Keyword(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.
Graduation Semester
2011-05
Permalink
http://hdl.handle.net/2142/24043
Copyright and License Information
Copyright 2011 Apratim Dhar

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