Temperature dependence of protein folding kinetics in living cells.
We measure the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) inside bone tissue cells as a function of temperature from 38 to 48 °C. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min. We find that this method yields improved thermal melts compared to heating a sample chamber or microscope stage. By comparing results for six cells with in vitro data, we show that the protein is stabilized by about 6 kJ/mole in the cytoplasm, but the temperature dependence of folding kinetics is similar to in vitro. The main difference is a slightly steeper temperature dependence of the folding rate in some cells that can be rationalized in terms of temperature-dependent crowding, local viscosity, or hydrophobicity. The observed rate coefficients can be fitted within measurement uncertainty by an effective two-state model, even though PGK folds by a multistate mechanism. We validate the effective two-state model with a three-state free energy landscape of PGK to illustrate that the effective fitting parameters can represent a more complex underlying free energy landscape.
Department of Physics, Chemical and Biomolecular Engineering, and Chemistry and Center for Biophysics and Computational Biology, University of Illinois, Urbana, IL 61801, USA.
Minghao
M
Guo
Yangfan
Y
Xu
Martin
M
Gruebele
D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007700:Kinetics; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding
D010735:Phosphoglycerate Kinase