Crowding and function reunite.

T he insides of cells are crowded. Fig. 1A is a dramatic representation of the fact that macromolecules occupy 30% of the cellular volume, with their concentrations reaching 300 g/L (1). For comparison, the concentration of protein in an egg white is only one third of that value. However, almost everything we know about biological macromolecules in solution comes from data acquired under conditions in which the concentration of macromolecules rarely exceeds 10 g/L. In PNAS, Dhar et al. (2) report the broadest study to date about the effects of macromolecular crowding on the properties of a globular protein. A powerful combination of experiment and theory is brought to bear here because the work is a collaboration between a team of experimentalists, the Gruebele group, and a team of theoreticians, the Cheung group. Their data run the gamut of crowding effects, covering folding kinetics, equilibrium stability, structure, and function. Before describing the system and the results, we consider what is expected when a globular protein in solution is crowded by other macromolecules. An elevator ride provides a simple analogy. If you are alone, it is easy to raise your wrist to eye level to check your watch. If the elevator is crowded, you are more likely to keep your hands to your sides. In terms of equilibrium, this is just a restatement of Le Chatelier’s principle: crowding favors species that require the least space. In the late 1950s, Ogston (3) suggested that volume exclusion by large molecules would affect equilibria. Three years later, Ogston and Phelps (4) proved it. Minton and Wilf (5) put the field on thermodynamic terra firma in terms of theory and experiment, and they coined the phrase “macromolecular crowding” in 1981. The most recent crowding paper to evoke intense interest among biochemists is from the groups of Wittung-Stafshede and Cheung, showing that crowding can deform the native state of a distinctly nonspherical globular protein, because a nonnative state takes up less space than its native state (6). A thorough and thought-provoking review by Elcock (7) of crowding research showcases the current state of the field. Despite early interest (5), more effort has been focused on crowding and protein stability than on crowding and protein function. Dhar et al. (2) bring function to the forefront again with their study of the enzyme phosphoglycerate kinase, which catalyzes the initial ATP-generating step of glycolysis. Most importantly, the enzyme is stunningly bilobal (Fig. 1B), which makes its open and unbound state distinctly noncompact. One substrate, ADP, binds an inner face, whereas the other substrate, 1,3-bisphosphoglycerate, binds the opposing inner face. Bringing the lobes together is thought to be involved in catalysis (8, 9). As such, crowding would be predicted to increase the enzyme’s activity. The authors tested this hypothesis by crowding a solution containing phosphoglycerate kinase with the highly soluble, uncharged, 70-kDa synthetic polymer Ficoll. The polymer is made by crosslinking sucrose molecules and is most well known for its ability to separate the components of blood serum. To detect changes in distance between the lobes, the Gruebele group covalently attached a different fluorescent label to each lobe and monitored Ficoll’s effect on the fluorescence [i.e., fluorescence resonance energy transfer (FRET)]. Cheung’s group focused on the computer simulations that provide molecular-level interpretations of the data. The simulations start with the crystal structure of the enzyme (PDB ID code 1QBG) and modeled Ficoll as a hard sphere with a radius of 5.5 nm. First, the effect of Ficoll on protein stability was measured by assessing the melting temperature. This task was accomplished by monitoring the FRET signal while raising the temperature. Like many proteins, this one denatures cooperatively, and the authors find that the midpoint of the transition increases with Ficoll concentration. It is well known that denatured states are less compact than native states; thus, the knee-jerk conclusion is that macromolecular crowding stabilizes the protein because the native state takes up less space. Not simply resting at this result, Dhar et al. (2) performed an important control to determine whether the macromolecular nature of Ficoll causes the stability increase. The fundamental component of Ficoll, sucrose, was used as a comparable small molecule crowder. The result was revealing; sucrose alone increases the melting temperature. Thus, the stabilization afforded by Ficoll is from favorable interactions between the polymer and the native protein (10) or a solvophobic effect (11) rather than a result of macromolecular crowding. Crowding effects on folding rates were examined by jumping the temperature and looking at relaxation of the FRET response. The interesting observation is that a minimum at 100 g/L is observed in a plot of the time constant for relaxation vs. Ficoll concentration. Results from simulations provide an explanation. The relaxation rate increase from 0 to 100 g/L is interpreted as decompaction of the denatured state ensemble. This interpretation stands in contrast to work recently described by the Gierasch group (12), suggesting a decrease in folding rates from crowding-induced collapse of the denatured state. The decreasing relaxation Fig. 1. (A) TheEscherichia coli cytoplasm,asmodeled by McGuffee and Elcock (1). (B) Open form of phosphoglycerate kinase (PDB ID code 1QPG), visualized with visual molecular dynamics (20).