In Vitro effects of Macromolecular Crowding on Protein Stability, Structure and Folding

Abstract

Proteins fold and function inside cell compartments that are full of other proteins, membranes and DNA. The crowded environment results in increased viscosity, excluded-volume effects and amplified opportunity for specific and non-specific inter-molecular interactions. These environmental factors are not accounted for in the mechanistic studies of protein folding and function that have been executed during the last decades. The question thus arises as for how these effects - present when polypeptides normally fold in vivo - modulate protein biophysical parameters? To take a step closer to understanding the in vivo scenario, we assess how crowded environments affect protein stability, structure and folding reactions in vitro. For this we use synthetic macromolecular crowding agents, which take up significant volume but do not interact with the target proteins, in combination with strategically selected proteins and a range of biophysical/spectroscopic methods. We have found that in the presence of macromolecular crowding in vitro, proteins become more thermodynamically stable (magnitude depends inversely on protein stability in buffer) and, protein-folded states may change both secondary structure content and overall shape. Finally, excluded volume effects may speed up folding kinetics and decrease the ruggedness of the folding energy landscape. Our findings demonstrate that excluded volume effects tune protein biophysical parameters: this is of mechanistic relevance since proteins have evolved to fold and function in crowded environments.