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.protein NMR | protein thermodynamics | protein folding | in-cell NMR C lassic theories about the effects of complex environments consider only hard-core repulsions (volume exclusion) and so predict entropy-driven protein stabilization (1-3). Here, we use the 7-kDa globular N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) as a model to test this idea in living cells. SH3 exists in a dynamic equilibrium between the folded state and the unfolded ensemble (4). This two-state behavior (5) is ideal for NMR-based studies of folding. Fluorine labeling (6) of its sole tryptophan leads to only two 19 F resonances (7): one from the folded state, the other from the unfolded ensemble (Fig. 1A). The area under each resonance is proportional to its population, ρ f and ρ u, respectively. These populations are used to quantify protein stability via the modified standard state free energy of unfolding,where R is the gas constant and T is the absolute temperature. Furthermore, the width at half height of each resonance is proportional to the transverse relaxation rate, which is an approximate measure of intermolecular interactions (8-10). Thus, this simple system yields both quantitative thermodynamic knowledge and information about interactions involving the folded state and the unfolded ensemble.To assess the enthalpic (ΔH°′ U ) and entropic (ΔS°′ U ) components, we measured the temperature dependence of ΔG°′ U .

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