Crowding-induced protein destabilization in the absence of soft attractions.

It is generally assumed that volume exclusion by macromolecular crowders universally stabilizes the native states of proteins and destabilization suggests soft attractions between crowders and protein. Here we show that proteins can be destabilized even by crowders that are purely repulsive. With a coarse-grained sequence-based model, we study the folding thermodynamics of two sequences with different native folds, a helical hairpin and a β-barrel, in a range of crowder volume fractions, φc. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with φc. Hence, for all φc>0, U is entropically disfavored relative to N. This entropy-centric view holds for the helical hairpin protein, which is stabilized under all crowded conditions as quantified by changes in either the folding midpoint temperature, Tm, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-φc conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at T<Tm, such that U is only weakly disfavored entropically by the crowders; and 2) a transient, compact, and relatively low-energy nonnative state that has a maximum population of only a few percent at φc=0, but increasing monotonically with φc. Overall, protein destabilization driven by hard-core effects appears possible when a compaction of U leads to even a modest population of compact nonnative states that are energetically competitive with N.