Walking across a sparsely occupied plaza, it shouldn’t take long to meet up with a person strolling through from the other side. Now, imagine crossing that same plaza filled with throngs of people. How much longer will one need to reach the other person?
A similar question is often asked by biochemists who study proteins. They can easily observe how long it takes two proteins to interact in a test tube solution. Test tubes are something like open plazas. The insides of cells, however, are extremely crowded spaces: To meet and interact, the proteins must make their way through a teeming mass of other macromolecules in the cell’s cytoplasm. In attempts to partially answer the question, scientists have tried adding other proteins and protein-like substances to their test tubes to simulate the crowd effect.
Yet the question remained open. Recently, Prof. Gideon Schreiber
, research student Yael Phillip and Dr. Vladimir Kiss of the Biological Chemistry Department decided to resolve the issue by developing a method to directly observe protein interactions in individual living cells. Their results
– an experimental first – appeared in the Proceedings of the National Academy of Sciences (PNAS)
To measure the interaction rate, the scientists needed a starting point. Creating that moment in time involved manipulating single cells into producing one of the proteins internally. The other protein was carefully injected into the cell with a microscopic needle as the measuring commenced. Both proteins were tagged with fluorescent molecules that produced a glow when an energy transfer took place, showing that binding was occurring between pairs of molecules.
Protein interaction in a cell. Lower left – light microscopy image shows the time from the injection of the second protein. Upper left – donor proteins appear in green. Upper right – as the proteins interact, energy is transferred to the acceptor proteins, causing them to glow red. Lower right – donor and acceptor dynamics combined
To their surprise, the scientists found that interaction rates for the proteins they tested were just a bit slower in a cell than they were in test tube solutions. Even when they mutated the proteins to make them faster or slower, the comparative rates did not differ by much.
Although they can’t yet be sure, Schreiber and his team think that, while counterintuitive, the crowd scenario could help explain why the protein interaction rates in living cells are faster than might have been expected in the thickly populated cytoplasm. “At first,” he says, “the crowding does slow the proteins down. But as they get closer to one another, the busy throng actually jostles them into meeting. These proteins don’t automatically recognize one another – they can bump into each other many times before an interaction takes place. Bouncing back and forth in the bustling mass of molecules increases the number of chance encounters between the two proteins, and thus the odds of interaction.”
On the one hand, says Schreiber, the new results provide strong evidence that the myriad protein experiments done in test tubes should give a good approximation of the true interaction rate. On the other, the method created in his lab is likely to advance the study of molecular interactions in their natural setting, inside living cells.
Schreiber: “This is the first time that anyone has managed to observe molecular interaction rates inside a single living cell. With the fluorescent imaging technology, we were literally able to see the interactions as they took place – we could determine not just the rate but the concentration of the different molecules over time as well. The team found that we could even focus on specific areas within the cell body. We are continuing to develop new experiments with the method, and other scientists have already expressed interest in using it to perform various biomolecular studies.”