Protein Stability and Folding Kinetics in the Nucleus and Endoplasmic Reticulum of Eucaryotic Cells

• Published in: Biophysical journal 2011-07, Vol.101 (2), p.421-430
• Main Authors: Dhar, A., Girdhar, K., Singh, D., Gelman, H., Ebbinghaus, S., Gruebele, M.
• Format: Article
• Language: English
• Subjects: Aqueous solutions; Cell Compartmentation; Cell Nucleus - enzymology; Cytoplasm; endoplasmic reticulum; Endoplasmic Reticulum - enzymology; energy transfer; Enzyme Stability; Eukaryotes; eukaryotic cells; Eukaryotic Cells - enzymology; Fluorescence Resonance Energy Transfer; Kinases; Kinetics; phosphoglycerate kinase; Phosphoglycerate Kinase - chemistry; Phosphoglycerate Kinase - metabolism; Protein; Protein Folding; Protein Transport; Saccharomyces cerevisiae - cytology; Saccharomyces cerevisiae - enzymology; Subcellular Fractions - enzymology; thermodynamics; Transition Temperature
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2011.05.071

We measure the stability and folding relaxation rate of phosphoglycerate kinase (PGK) Förster resonance energy transfer (FRET) constructs localized in the nucleus or in the endoplasmic reticulum (ER) of eukaryotic cells. PGK has a more compact native state in the cellular compartments than in aqueous solution. Its native FRET signature is similar to that previously observed in a carbohydrate-crowding matrix, consistent with crowding being responsible for the compact native state of PGK in the cell. PGK folds through multiple states in vitro, but its folding kinetics is more two-state-like in the ER, so the folding mechanism can be modified by intracellular compartments. The nucleus increases PGK stability and folding rate over the cytoplasm and ER, even though the density of crowders in the nucleus is no greater than in the ER or cytoplasm. Nuclear folding kinetics (and to a lesser extent, thermodynamics) vary less from cell to cell than in the cytoplasm or ER, indicating a more homogeneous crowding and chemical environment in the nucleus.