BIOPHYSICS:

What's macromolecular crowding, and why should anyone care about it? An obvious yet underappreciated characteristic of living cells is that they have lots and lots of protein molecules in them, often in excess of 10% of their dry weight.

In other words, the cell interior is very crowded with macromolecules (proteins). The large numbers of protein molecules "swimming" around within the cell, and the large size of proteins, come together to dramatically reduce the volume available for proteins to move around the cell interior.

Stated differently, the volume occupied by the protein molecules is significant relative to the surrounding unoccupied water solution. This is contrary to the common chemical assumption of negligible molecular volume occupancy.

This is not a trivial academic curiosity; it has profound implications for the very nature of life itself. According to a vast set of experimental and theoretical studies, macromolecular crowding impacts protein folding and self-assembly, enzymatic reaction rates, biochemical signaling, and many other critical intracellular phenomena.

Through aqueous (water-based) phase separation, macromolecular crowding may also provide a means of spontaneous intracellular organization. Full disclosure: This was the highlight of my research in graduate school, and I'm clearly biased as to how cool the topic is.

Much of our biochemical knowledge needs to be questioned.

What does all of this mean? The proper function of a cell critically depends upon the obvious, yet underappreciated, physical fact that the cell interior is crowded with protein molecules.

Consequently, macromolecular crowding cannot be ignored when unraveling the structure and function of proteins. Thus, it has great ramifications for intracellular physiology, and the biomolecular basis of life.

However, most studies of the structure and function of proteins are performed in dilute water solution. As discussed, this is very different from a protein's native (in-cell), crowded environment.

Therefore, much of the research on intracellular physiology is based on a fundamentally false premise, i.e. that the cell interior is not crowded. It stands to reason that much of what we "know" about the biomolecular basis of life needs to be updated.

A new approach is needed.

Where can we begin this journey of revision? For starters, when feasible, scientists can start performing their biochemical experiments in crowded (concentrated) solutions, a concept that is gaining increasing awareness and acceptance.

Theoretical biochemical investigations also need to include the effect of macromolecular crowding; many such investigations have been published. However, the limitations of these studies to date are that they have yielded nonquantitative results or have been computationally intensive, both of which limit the level of biochemical detail that can be unraveled.

Nikolay Dokholyan (University of North Carolina at Chapel Hill, United States) and coworkers have addressed this issue. Their simplified approximation of macromolecular crowding yields quantitative data that agrees with experiments and computationally-intensive theories of protein stability to unfolding.

Basic principles of the scientists' model.

The scientists' theoretical model of macromolecular crowding, in respect to protein folding, reduces the number of variables that could, in principle, be under consideration. This renders the model solvable, yet as they demonstrate, their approach does not sacrifice the model's quantitative power.

Specifically, they treat a protein as two rigid spheres (a rough approximation of many proteins). The spheres interact both directly and indirectly.

Direct, short-range interactions refer to hydrogen bonding, electrostatic interactions, and hydrophobic interactions between the amino acids (protein subunits) comprising the protein. They can be disrupted once they reach a certain level of energy (e.g. through disruption by urea, a molecule often used by biochemists to initiate protein unfolding).

Indirect, long-range interactions refer to strong covalent bonds (equal to somewhat equal sharing of electrons) between the amino acid linkages. They are constrained by the covalent bonds' radius of gyration (a measure of the bond length) upon protein unfolding.

Both types of interactions in the model are designed to follow experimentally-determined predictions for ribonuclease A, a two-layer protein which has been studied for many years. The scientists' model is clearly primitive, yet it nevertheless captures certain aspects of protein folding while enabling in-depth computation.

Model predictions and comparison to experiments.

Using their model, the scientists predict that the resistance of a hypothetical protein to urea-induced unfolding strongly depends upon the size of the protein in relation to the crowding agent (i.e. other proteins or additional copies of the same protein). Specifically, the resistance increases as the crowding agent size decreases.

The scientists' predictions are generally in quantitative agreement with the results of actual experiments, and are a standard outcome of macromolecular crowding simulations. They are also in close agreement with much more intensive and detailed theoretical models, validating the use of the former for quantitative theoretical investigations of macromolecular crowding.

The effect of crowding agent size should be even greater for real proteins than is predicted by this study. Real proteins in a living cell gain much more contact area with crowding agents when they unfold than that predicted here (proteins are nanometer-scale entities comprised of far more than two subunits).

Overall evaluation.

Experiments are the best method of teasing apart the inner workings of a living cell. When experiments are impossible or infeasible, theory is the next best thing, but theoretical models must be based on reasonable physical principles to be useful.

Dokholyan and coworkers have developed a primitive, yet highly useful, theoretical model for the effects of macromolecular crowding on protein folding stability. They have found that their model generally agrees with experimental results obtained for the protein ribonuclease A.

They have also found that their theoretical model agrees well with models that are far more involved and time-consuming. This development should be very useful for probing the consequences of macromolecular crowding on cellular physiology, and consquently on a fundamental physical property governing the very nature of life itself.

for more information:
Tsao, D., Minton, A. P., & Dokholyan, N. V. (2010). A Didactic Model of Macromolecular Crowding Effects on Protein Folding PLoS ONE, 5 (8) DOI: 10.1371/journal.pone.0011936