Protein Structure, Stability and Folding in the Cell - in vitro Biophysical Approaches

Folding processes of simple proteins have been studied for years in test tubes. However, many proteins in the cells are more complex: some bind cofactors and others interact with other polypeptides in order to form their functional units. To obtain mechanistic information of the folding reactions of such proteins, we combine protein engineering in strategic model systems (e.g., a/b flavodoxin, b-barrel azurin, all-a VlsE) with a range of biophysical methods (e.g., circular dichroism, stopped-flow mixing, calorimetry). To take a step closer to the in vivo scenario, we assess how the crowdedness of the cell milieu affects protein biophysical parameters using synthetic macromolecular crowding agents (e.g., Ficoll, dextran) that take up significant volume but do not interact with the targets or have interfering spectroscopic signals. We have found that in the presence of macromolecular crowding in vitro, proteins can fold faster, become more thermodynamically stable and, surprisingly, the folded forms may change in terms of both secondary structure content and overall shape. Our discoveries imply that Nature may use excluded volume effects as a tool to tune protein biophysical parameters, and thereby function, in vivo.