Understanding the Effectiveness of Synthetic Crowding Agents

Abstract

In vitro studies on the structure and stability of macromolecules are typically performed using very dilute solutions. However, the total intracellular concentration of macromolecules is very high, resulting in an in vivo environment that is significantly crowded. Prior studies have proven that the nonspecific interactions that occur between individual macromolecules and their crowded surroundings have a significant effect on biochemical rates and equilibira. In other words, the mechanisms under which a protein functions in a living cell may be quite different from the conditions under which a protein is studied by biochemist in the laboratory. To gain a better understanding of the phenomenon of macromolecular crowding, researchers have begun to utilize synthetic crowding agents such as ficoll, dextran, and PEG to recreate the in vivo environment. Experiments are conducted to understand the properties of proteins in such conditions with the belief that these synthetic crowding agents are able to adequately mimic the intracellular environment with its multiple components of lipids, carbohydrates, nucleic acids, and proteins. These crowding agents are thought to serve as inert compounds that have no interaction with the protein in question. This study has investigated the ability of synthetic crowding agents to produce a cellular environment that is similar to that of the actual cell. The thermal denaturations and NMR spectra of lysozyme and fibroblast growth factor (hFGF) were tested in the presence of various synthetic crowding agents. This was compared with the thermal denaturation and NMR spectra of these same proteins when placed in higher concentrations of themselves. The results indicate that synthetic crowding agents are not effective in mimicking the cellular environment. With these results, the understanding of protein study in the laboratory can be furthered as techniques to create a life like laboratory environment are refined.