In-cell thermodynamics and a new role for protein surfaces

Understanding protein thermodynamics as it occurs inside cells is a fundamental goal of biophysics, and, from a practical point of view, will facilitate the design and improvement of protein-based drugs and catalysts. By measuring the temperature dependence of protein stability inside Escherichia coli cells, we show, contrary to predictions, that proteins are not necessarily stabilized inside cells compared with buffer alone. We also show that crowding-induced charge–charge interactions slow folding because of preferential interactions with the unfolded ensemble, and reducing these interactions increases protein stability

Protein Fibrillation under Crowded Conditions

Protein amyloid fibrils have widespread implications for human health. Over the last twenty years, fibrillation has been studied using a variety of crowding agents to mimic the packed interior of cells or to probe the mechanisms and pathways of the process. We tabulate and review these results by considering three classes of crowding agent: synthetic polymers, osmolytes and other small molecules, and globular proteins. While some patterns are observable for certain crowding agents, the results are highly variable and often depend on the specific pairing of crowder and fibrillating protein

In-cell thermodynamics and a new role for protein surfaces

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