Intracellular Measurement of Macromolecular Crowding by a Genetically-Encoded Indicator

Abstract

Intracellular environment is overcrowded with enormous number of various macromolecules such as proteins. For example, the total concentration of macromolecules in Escherichia coli (E. coli) cells is known to be about 350 mg/ml. This molecular crowding reduces the substantial space for molecular diffusion which must affect the biological phenomena including enzymatic reaction, protein-protein interaction, protein-DNA interaction, and protein folding. Since the crowding conditions are a ubiquitous feature in not only prokaryotic cells but also eukaryotic cells, biophysical and biochemical measurements in vitro using cell extracts maintaining natural cellular contents are one of essential approaches. However, this type of experiments inevitably lost a spatio-temporal information of molecular crowding in living cells. To investigate the crowding effect on biomolecular functions in living cells, we have been trying to develop a genetically-encoded fluorescent indicator for molecular crowding. During the course of engineering in a yellow variant of green fluorescent protein from Aequorea Victoria, we found that a variant inserted glycine residue just before Thr145 (YFP1G) changed in its fluorescence intensity according to the protein concentration while cyan variant of fluorescent protein (CFP) did not. Then, we fused CFP with YFP1G (CFP-YFP1G) to make a ratiometric indicator. The emission ratio (YFP/CFP) of CFP-YFP1G was decreased by increasing in protein concentration.When expressed in E. coli, fluorescence signal of CFP-YFP1G was dramatically changed in synchronization with the cell cycle, i.e. protein concentration increased with cell growth but decreased after cell division. In addition, we obtained a similar result in mammalian cells. This CFP-YFP1G will become an indispensable tool to figure out relationship between biomolecular functions and macromolecular crowding.