Loss of folding cooperativity as a function of crowder size. (A) At constant fractional volume, both the specific heat and folding cooperativity of SH3 domain decrease with respect to decreasing crowder diameter. Legend: () 5 Å, ()10Å, () 15Å, (-·-) 20 Å crowder diameter. Gray regions indicate the estimated error associated with each curve. (B) Comparison of the degree of cooperativity for SH3 domain under various crowding conditions. The top X-axis corresponds to the open circles plotted and represent the set of simulations at constant fractional volume. For comparison we have plotted the degree of cooperativity as a function of crowder concentration at a constant diameter of 10 Å, measured on the bottom X-axis and denoted by closed squares. Dashed lines are plotted to aid in identifying the trends between the two sets. (C) Energy histogram plotted for SH3 domain under different crowding diameters at constant fractional volume. Two-state folding kinetics are represented by the folded state on the left and the unfolded state on the right. The diminishing and shifting peak representative of the unfolded state is indicative of effective compacting obtained by smaller crowders. Legend: () No crowders, () 20 Å, ()15 Å, (-·-) 5 Å.