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, ${\phi }_{\text{c}}$. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with ${\phi }_{\text{c}}$. Hence, for all ${\phi }_{\text{c}}>0$, 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, $T_{\text{m}}$, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-${\phi }_{\text{c}}$ conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at $T<T_{\text{m}}$, 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 ${\phi }_{\text{c}}=0$, but increasing monotonically with ${\phi }_{\text{c}}$. 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.

通常认为，大分子拥挤剂的体积排除普遍稳定了蛋白质的天然状态，而不稳定表明拥挤剂和蛋白质之间的软吸引力。在这里，我们表明，即使是纯粹令人排斥的拥挤者也可能使蛋白质不稳定。通过基于粗粒度序列的模型，我们研究了具有不同原生折叠的两个序列（螺旋发夹和 β 桶）在一系列拥挤体积分数中的折叠热力学，${\phi }_{\text{C}}$。我们发现，原始状态 N 在拥挤条件下保持结构不变，而展开状态 U 的大小随着${\phi }_{\text{C}}$。因此，对于所有${\phi }_{\text{C}}>0$，U 相对于 N 来说是熵不利的。这种以熵为中心的观点适用于螺旋发夹蛋白，它在所有拥挤条件下都是稳定的，通过折叠中点温度的变化进行量化，${\mathrm{时间}}_{\text{米}}$，或折叠自由能。然而，我们发现 β-桶蛋白在低T、低 T条件下不稳定。${\phi }_{\text{C}}$状况。这种不稳定可以从其折叠的两个特征来理解：1）相对紧凑的U形$\mathrm{时间}<{\mathrm{时间}}_{\text{米}}$，使得 U 仅受到拥挤者微弱的熵不利；2) 一种瞬态、紧凑且能量相对较低的非原生态，其最大种群数量仅为百分之几${\phi }_{\text{C}}=0$，但单调增加${\phi }_{\text{C}}$。总体而言，当 U 的压缩甚至导致与 N 激烈竞争的少量紧凑非本地状态时，由核心效应驱动的蛋白质不稳定似乎是可能的。