Protein folding in crowded environments and living cells

protein folding;live cells;in vivo;macromolecular crowding;fast relaxation imaging;Förster Resonance Energy Transfer (FRET);folding kinetics;thermal denaturation

Dhar, Apratim

关键词: protein folding;    live cells;    in vivo;    macromolecular crowding;    fast relaxation imaging;    Förster Resonance Energy Transfer (FRET);    folding kinetics;    thermal denaturation;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/24043/Dhar_Apratim.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

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.