title: Protein stability and folding kinetics in the nucleus and endoplasmic reticulum of eucaryotic cells.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
authors.forename: A
authors.initials: A
authors.lastname: Dhar
authors.forename: K
authors.initials: K
authors.lastname: Girdhar
authors.forename: D
authors.initials: D
authors.lastname: Singh
authors.forename: H
authors.initials: H
authors.lastname: Gelman
authors.forename: S
authors.initials: S
authors.lastname: Ebbinghaus
authors.forename: M
authors.initials: M
authors.lastname: Gruebele
mesh_terms: D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000201:enzymology*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004795:Enzyme Stability; D005057:Eukaryotic Cells / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D044366:Transition Temperature
chemical_list: D010735:Phosphoglycerate Kinase