Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St Johns, Newfoundland and Labrador, Canada.
Department of Physics and Physical Oceanography, Memorial University of Newfoundland, St Johns, Newfoundland and Labrador, Canada.
Biophys J. 2022 Jul 5;121(13):2503-2513. doi: 10.1016/j.bpj.2022.06.005. Epub 2022 Jun 7.
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, φ. We find that the native state, N, remains structurally unchanged under crowded conditions, while the size of the unfolded state, U, decreases monotonically with φ. Hence, for all φ>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, or the free energy of folding. We find, however, that the β-barrel protein is destabilized under low-T, low-φ conditions. This destabilization can be understood from two characteristics of its folding: 1) a relatively compact U at T<T, 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 φ=0, but increasing monotonically with φ. 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，大分子拥挤剂通过体积排阻普遍稳定蛋白质的天然状态，而使蛋白质不稳定则表明拥挤剂与蛋白质之间存在软吸引。在这里，我们表明，即使是纯粹排斥的拥挤剂也可以使蛋白质不稳定。我们使用基于序列的粗粒度模型，研究了两种具有不同天然折叠的序列（发夹螺旋和β-桶）在一系列拥挤剂体积分数φ下的折叠热力学。我们发现，在拥挤条件下，天然状态 N 保持结构不变，而展开状态 U 的大小则单调减小随着 φ 的增加而增加。因此，对于所有 φ>0，U 相对于 N 在熵上是不利的。这种以熵为中心的观点适用于发夹螺旋蛋白，因为根据折叠中点温度 T 或折叠自由能的变化，它在所有拥挤条件下都得到了稳定。然而，我们发现β-桶蛋白在低 T、低 φ 条件下是不稳定的。这种失稳可以从其折叠的两个特征来理解：1）在 T<T 时 U 相对紧凑，使得 U 仅受到拥挤剂的微弱熵不利影响；2）一个瞬态、紧凑且相对低能量的非天然状态，其在 φ=0 时的最大种群只有百分之几，但随着 φ 的增加而单调增加。总体而言，当 U 的紧凑化导致与 N 具有竞争力的紧凑非天然状态的种群甚至略有增加时，由硬芯效应驱动的蛋白质失稳似乎是可能的。
