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.

Residue level quantification of protein stability in living cells

“…Using a k cl of 10 3 s −1 (30) and the average value of −RT lnðk obs =k int Þ for the 17 residues quantified in cells, the cytoplasm would have to decrease k op 10 2 -10 3 -fold and decrease k cl by an order of magnitude compared with dilute solution to force exchange into the regime where k cl is rate determining. Such drastic effects are unlikely and have never been observed in cells (5,6,8,63). In summary, the data are consistent with the assumption that we are measuring free energies of opening in cells.…”

“…Although the folding kinetics (5,6,8,51,63) and equilibrium thermodynamic stability (5-9) of globular proteins can be influenced by crowding, their tertiary structures should remain unchanged (51,54,64) because the packing densities of globular proteins approximate those for ideal packing of hard spheres (65). As discussed above, the ability to overlay the in-cell spectrum with that from dilute solution is consistent with this expectation.…”

“…However, this idea may be too simple, as indicated by our observation on the effect of BSA and the temperature dependence of crowding discussed by Zhou (71). Another complication is the role the cell has in modulating stability via compartmentalization, as highlighted by Gruebele and coworkers (5,9). Although the present study enriches our knowledge of the forces stabilizing proteins under native conditions, more studies are necessary to bring about a comprehensive understanding of the effects of cellular crowding on protein stability.…”

“…Nevertheless, most biochemical and biophysical studies are conducted under dilute (<10 g/L macromolecules) conditions. Here, we augment the small but growing list of reports probing the equilibrium thermodynamic stability of proteins in living cells (5)(6)(7)(8)(9), and provide, to our knowledge, the first measurement of residue-level stability under nonperturbing conditions.…”

Soft interactions and crowding

“…The rates increased, suggesting destabilization of the protein. The Gruebele group studied the stability of the test protein phosphoglycerate kinase in human cell lines by using fluorescence spectroscopy (Ebbinghaus et al 2010;Dhar et al 2011;Guo et al 2012). Their main conclusion was that the folding kinetics, stability, and dynamics are not only affected by crowding but also by the environment in different cellular compartments.…”