In-cell thermodynamics and a new role for protein surfaces

Abstract

There is abundant, physiologically relevant knowledge about protein cores; they are hydrophobic, exquisitely well packed, and nearly all hydrogen bonds are satisfied. An equivalent understanding of protein surfaces has remained elusive because proteins are almost exclusively studied in vitro in simple aqueous solutions. Here, we establish the essential physiological roles played by protein surfaces by measuring the equilibrium thermodynamics and kinetics of protein folding in the complex environment of living Escherichia coli cells, and under physiologically relevant in vitro conditions. Fluorine NMR data on the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) show that charge-charge interactions are fundamental to protein stability and folding kinetics in cells. Our results contradict predictions from accepted theories of macromolecular crowding and show that cosolutes commonly used to mimic the cellular interior do not yield physiologically relevant information. As such, we provide the foundation for a complete picture of protein chemistry in cells.

Keywords: in-cell NMR; protein NMR; protein folding; protein thermodynamics.

Fig. 1.

Fluorine spectra acquired at 298 K, in buffer (A) and cells (B). The blue trace is from the postexperiment supernatant and shows that the red spectrum arises from protein inside cells. Stability curves (C) in buffer (black), in cells (red and green), and in 100 g/L urea (magenta). In-cell metabolite correction and analysis of uncertainties are discussed in Results and Discussion and Materials and Methods, respectively. Shaded regions are 95% confidence intervals. Error bars for buffer are smaller than the labels and represent the SD of three trials. Error bars for the in-cell data at 273, 298, and 313 K represent the SD of three trials. Stability in buffer (black) and solutions of 100 g/L BSA (blue) and lysozyme (red) at different pH values (D–F). The curve for buffer from C is reproduced in D. The net charges on SH3, BSA, and lysozyme (based on sequence) are shown. Error bars (298 K) represent the SD from three trials. Appearance of new resonances in the pH 3 BSA sample prevented extraction of thermodynamic parameters.

Fig. 2.

Correcting for in-cell and supernatant metabolite. (A) In-cell ¹⁹F spectrum showing integration regions for the folded (F_IC) and unfolded/metabolite (U_IC) peaks at 298 K. (B) Supernatant spectrum showing integration region for leaked metabolite (S). (C) Spectrum of lysed and diluted in-cell sample. (We always use the spectrum of the lysate from the corresponding in-cell sample.) A peak for the folded state (F_lysate), unfolded ensemble (U_lysate), and a metabolite (M_lysate) are observed. (D–F) Spectra at 318 K.

Fig. 3.

Tumbling and folding. Symbol size reflects the uncertainty. (A) Resonance broadening. (B) Tumbling times. (C) Folding rates (100 g/L lysozyme, BSA and urea, and 300 g/L Ficoll, pH 7.2, 298 K).

Fig. 4.

Synthetic polymers and their monomers. (A) Glucose and dextran, (B) sucrose and Ficoll (all at 300 g/L), (C) ethylene glycol, 8 kDa PEG, and 35 kDa PEG (all at 200 g/L) stabilize the SH3 domain. Buffer (black) curve is reproduced from Fig. 1C. Error bars for the 298-K data are the SD of three trials.