The simulations presented here are unique in that they are the first to utilize the replica-exchange method in conjunction with discrete molecular dynamics to study the effects of macromolecular crowding. The combination of these two algorithms has allowed us to explore a large sample of configurations enabling us to conduct thermodynamic analyses.
Modeling proteins with a Gō potential is an effective way to explore conformational space for fast-folding two-state proteins9,38
and has been utilized extensively to study protein dynamics including under macromolecular crowding conditions.32,39
With the explicit modeling of crowders as hard spheres and proteins at a residue-specific level using a two-bead Gō model, our simulation results are in line with those previously published.4
In accordance with theory, increasing the fractional volume occupied by crowders within a system (either by concentration or size) compresses a polypeptide chain into a compact state thereby stabilizing the native state of a protein. Both our results and those previously reported by others suggest that macromolecular crowding can vary a protein's ensemble of states in a non-monotonic fashion. Using an off-lattice model, Cheung et al
. found that the refolding rates of the WW domain increased nonmonotonically with increasing fractional volumes occupied by the crowders.40
By altering either the concentration or the width of crowders, we observe a nonmonotonic shift in protein equilibria towards the native state ().
Our simulations are also able to recapitulate the biophysical phenomena where smaller crowders reduces the void volume near the proximity of a protein and consequently stabilizes a compact conformation.37
We find that there is not only a shift in equilibria to favor the native state, but also compacted unfolded states under more aggressive crowding conditions. The shifts seen primarily in the unfolded region are similar to those reported under conditions of macromolecular confinenment.10
Overall, these crowding effects become more prominent for polypeptide chains of higher sequence length.
The most surprising finding within our set of simulations is that macromolecular crowding induces a loss of cooperativity in folding. This effect is observed irrespective of how macromolecular crowding is achieved. The implications of specific heat peak broadening are most easily understood in terms of two-state folding kinetics. We have simulated several proteins, in which most are known to exhibit two-state folding dynamics. Trp-cage has been characterized as a two-state folding protein both experimentally41
The SH3 domain has also been shown to follow two-state folding kinetics in both computation43
and through experiments,44
consistent with our current set of simulations that SH3 domain (). The villin headpiece folding pathway has also been demonstrated to be second-order.45,46
It is unclear whether or not the α-domain of hemoglobin follows a two-state folding model though the single sharp transition in the specific heat of folding suggests it does. Nonetheless, we have limited our cooperativity analyses to SH3 domain and Trp-cage.
The Gō model itself does not include actual physicochemical interactions. Rather, the Gō potential is based on the probability of residues being in close proximity within a protein's structure. Hence loss of folding cooperativity may potentially be an artifact due to the coarse-graining of our protein models or the use of a Gō potential. To test the robustness of our findings, we conducted all-atom simulations of Trp-cage using a force field that includes van der Waals packing, solvation, and environment-dependent hydrogen bond interactions. We have chosen Trp-cage since the protein is small enough to ensure adequate sampling of its conformational space despite the increase in atomic resolution. Our results with the all-atom model are consistent with those obtained using the two-bead Gō model ( and ). As the concentration of crowders increase, the two-state folding cooperativity decreases as represented by peak broadening.
Loss of folding cooperativity presents an interesting scenario. Macromolecular crowding effects have been mostly considered a beneficial stabilizing effect for protein folding when regarded as a purely volume exclusion effect. However, the loss of cooperative folding reveals a detrimental consequence of macromolecular crowding. By reducing the two-state folding properties of a protein, the probability of folding into a metastable/alternative state increases. There are biological implications for such behavior since the existence of non-native structures may promote the formation of potentially toxic protein aggregates. High crowder concentrations may also potentially trap proteins into structural intermediates that are present within the folding pathway.
Loss of folding cooperativity leads to alternative structures since crowders may force the protein to adopt a compact state regardless whether it is the native conformation or not. The idea that crowders may induce alternative states has been a topic of recent discussion.47
Formation of an alternative structure due to macromolecular crowding was recently discovered experimentally for the VlsE protein found in Borrelia burgdorferi
. The authors found that if the protein is denatured then refolded under the presence of high crowder concentrations, a change in the overall topology and secondary structure is observed. The conformational change found in this particular protein is biologically significant since crowding may be a method in which antibodies are generated against the bacteria by exposing key residues in its alternative state. Thus macromolecular crowding may modulate changes in conformations by trapping proteins in metastable states.
The Gō model has been previously employed in several studies to examine the cooperativity of folding for numerous proteins.32,48,49
Although it is successful in describing the cooperativity of two-state folders as well as identifying key intermediates in higher-order protein folding, the Gō model is biased toward the native state.38,50
It is possible to find novel alternative structures, but the enthalpy of those states will be higher by default since the native structure in the Gō model is assigned the lowest energy state. Additionally, the native state bias imposes restrictions on the sampling of non-native structures and thus these alternate structures will occur at a lower probability. More sophisticated force fields are necessary in order to thoroughly characterize alternative structures akin to the previous VlsE example.