Adenosine triphosphate (ATP) is the primary energy molecule of most cells. Without ATP, most cells would not survive for long.
One of the essential functions of the enzyme phosphoglycerate kinase is to synthesize ATP. This particular synthesis is a bit of a puzzle, and is thought to occur via two subunits of the enzyme (somehow) coming together like a hinge.
This hypothesis is largely based on experiments performed in dilute solution, and on crystal structures of the enzyme. These experiments are in turn based on a fundamental scientific misunderstanding, namely that the cell interior is not crowded.
What is intracellular crowding?
The interior of cells is crowded with proteins (i.e. "macromolecules"). This is a nontrivial observation, with far-ranging biochemical consequences.
Imagine a protein in a solution of small molecules. Even if the concentration of small molecules is very high, the volume occupied by these small molecules is still negligible, meaning that the volume available for the protein to move around (e.g. change its shape to execute a biochemical reaction) hasn't been significantly altered.
Now imagine a protein in a concentrated solution of very large molecules (e.g. other proteins). Proteins are rather large entities (roughly 5 nanometers in diameter on average), relative to most molecules.
The volume occupied by the extraneous proteins is very large. This means that the volume available for the protein of interest to move around is considerably reduced.
In other words, the common chemical assumption of negligible molecular volume occupancy does not hold for the cell interior. As discussed next, this has profound biochemical consequences.
The comparison is analogous to dancing within a room with twenty pennies in it, relative to within a room with twenty boulders in it. The pennies wouldn't affect your dancing, but you'd be constantly bumping into the boulders, limiting the moves you could perform.
Intracellular crowding: Possible implications for phosphoglycerate kinase.
Intracellular crowding has many dramatic effects on protein (e.g. enzyme) conformation, assembly, and function, both limiting and facilitating what proteins can do (e.g. the biochemical reactions the proteins can perform). It may even (at least partially) explain how the cell interior spontaneously organizes itself into a functional architecture.
It's reasonable to suspect that intracellular protein crowding has many effects on the architecture and function of phosphoglycerate kinase (remember that this is a critical intracellular enzyme), as yet unknown because scientists have yet to study the enzyme in a sufficiently realistic analog of its native environment (i.e. the cell interior). Martin Gruebele, Margaret Cheung (University of Illinois and University of Houston, both in the United States) and coworkers have carried out such a relevant study.
They have found that crowding fundamentally alters the architecture of phosphoglycerate kinase. Crowding spontaneously brings together the two enzymatic subunits critical to ATP synthesis, and dramatically speeds up the enzyme's reaction rate, relative to within dilute solution.
Crowding effects on phosphoglycerate kinase.
The scientists first found, via fluorescence, that the two previously-discussed subunits of the enzyme phosphoglycerate kinase come closer together when the enzyme is mixed with increasing Ficoll volume fraction (a measure of concentration, i.e. how much of the solution volume is taken up by a molecule). Ficoll is a crowding agent; specifically, a sucrose polymer, i.e. a primitive mimic of a "background protein."
Crowding effects are initially minimal. With more Ficoll, the enzmye subunits come progressively closer together until at least a Ficoll volume fraction of approximately 20%.
Minimal effects were seen in analogous experiments with sucrose (a small sugar molecule) in the place of the crowding agent Ficoll (a large carbohydrate polymer). Crowding agent volume fraction had minimal effect on inherent fluorescence, and little effect on protein stability to heat-induced denaturation (less than 2°C).
Thus, crowding is responsbile for the structural effects the scientists observed. Computer simulations back up these experimental results.
These computer simulations suggest that the most stable conformation of phosphoglyerate kinase at high crowding agent volume fractions nearly halves the distance between the two enzyme subunits critical to ATP synthesis (presumably lowering the energy necessary to perform the reaction; making it "easier" to perform), relative to the distance observed in dilute solution. Does crowding have functional implications for the enzyme?
Most impressively, increasing Ficoll (crowding agent) volume fraction dramatically increased the speed at which the enzmye phosphoglycerate kinase produces the energy molecule ATP. This is very interesting, because an increasing Ficoll volume fraction increases the viscosity of the solution, which would ordinarily be expected to slow down the reaction.
At a crowding agent volume fraction of approximately 13%, the speed of the enzymatic reaction increased by a factor of over 15 (taking into consideration reduced molecular diffusion in high viscosity solutions), relative to experiments performed in dilute solution. Analogous experiments with sucrose in the place of Ficoll simply reduced enzymatic reaction speed, due to increased viscosity without crowding effects.
Maximum enzyme folding rate is at a crowding agent volume fraction of roughly 13%. In other words, crowding may enhance enzymatic function to a point, after which higher concentrations may disrupt enzyme function (due to viscosity effects).
Implications and further experiments.
The enzyme phosphoglycerate kinase may already be in the proper shape to execute its functions when it is in the cell interior. Therefore, the hinge hypothesis of how it synthesizes ATP (energy molecules) may be irrelevant.
Put simply, protein crowding in the cell interior may dramatically simplify an important biochemical question. What are and might be in the scientists' future plans?
Future research may include the effect of polydispersity on crowding agent effects. In other words, some proteins are bigger than others, and this variation in size may influence the biochemical effect of crowding.
The scientists note that these results with phosphoglycerate kinase may translate to other multisubunit proteins. The only way to conclusively test this hypothesis is to perform further protein crowding experiments.
An enormous pile of experimental and theoretical work, going back at least three decades, shows that protein crowding in the cell interior cannot be ignored if one wishes to understand how living cells execute their functions. A proper understanding of how cells work in normal health is necessary to understand how cells may misfunction as a consequence of a disease.
It's long past time for the average cell biologist and biochemist to appreciate the importance of intracellular protein crowding, and to incorporate its effects in their experiments whenever possible. The alternative may be to fundamentally misunderstand the biochemical basis of life.
NOTE: The scientists' research was funded by the National Science Foundation and the von Humboldt Foundation. Additional resources were provided by the Texas Learning and Computation Center, the University of Houston, and the Center for the Physics of Living Cells (supported by the National Science Foundation).
Dhar, A., Samiotakis, A., Ebbinghaus, S., Nienhaus, L., Homouz, D., Gruebele, M., & Cheung, M. S. (2010). Structure, function, and folding of phosphoglycerate kinase are strongly perturbed by macromolecular crowding Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1006760107