Effects of Macromolecular Crowding on Protein Biophysics

Abstract

Proteins fold and function inside cells which are environments very different from that of a dilute buffer solution most often used in in vitro experiments. The cell compartments are full of other proteins, membranes and DNA; it is estimated that up to 40 % of the available volume in a cell can be occupied by other biomolecules. 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 biophysics? To take a step closer to understanding the in vivo scenario, we here assess how crowded environments affect protein biophysical properties. 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. For a protein with a complex folding mechanism involving an off-path intermediate, excluded volume effects make the folding energy landscape smoother (i.e., less misfolding) than in buffer. 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.