Protein folding stability and dynamics imaged in a living cell

Abstract

Biomolecular dynamics and stability are predominantly investigated in vitro and extrapolated to explain function in the living cell. We present fast relaxation imaging (FreI), which combines fluorescence microscopy and temperature jumps to probe biomolecular dynamics and stability inside a single living cell with high spatiotemporal resolution. We demonstrated the method by measuring the reversible fast folding kinetics as well as folding thermodynamics of a fluorescence resonance energy transfer (FRET) probe-labeled phosphoglycerate kinase construct in two human cell lines. Comparison with in vitro experiments at 23-49 degrees C showed that the cell environment influences protein stability and folding rate. FReI should also be applicable to the study of protein-protein interactions and heat-shock responses as well as to comparative studies of cell populations or whole organisms.

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.…”

“…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.…”

Protein-binding assays in biological liquids using microscale thermophoresis

“…This correction did not include possible binding-dependent changes in the temperature response of the molecule. Slow conformational variations were recently reported by Ebbinghaus et al 36 and can lead to an uncertainty when determining the exact depletion, but does not effect the precision of the analysis. The relative change in concentration was normalized to the saturation value, at which all molecules were in the bound state.…”

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.…”