Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, United States.
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, United States.
Biochemistry. 2021 Jan 26;60(3):170-181. doi: 10.1021/acs.biochem.0c00720. Epub 2021 Jan 12.
In addition to encoding the tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determine the speed of folding. How these kinetic barriers are encoded is not well understood. Here, we use evolutionary sequence variation in the α-lytic protease (αLP) protein family to probe the relationship between sequence and energy landscape. αLP has an unusual energy landscape: the native state of αLP is not the most thermodynamically favored conformation and, instead, remains folded due to a large kinetic barrier preventing unfolding. To fold, αLP utilizes an N-terminal pro region similar in size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant time scale. Without the pro region, αLP folds on the order of millennia. A phylogenetic search uncovers αLP homologs with a wide range of pro region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro region size. By studying homologs naturally lacking a pro region, we demonstrate they can be thermodynamically stable, fold much faster than αLP, yet retain the same fold as αLP. Key amino acids thought to contribute to αLP's extreme kinetic stability are lost in these homologs, supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on the protein sequence.
除了编码三级折叠和稳定性外，蛋白质的一级序列还编码折叠轨迹和动力学障碍，这些轨迹和障碍决定了折叠的速度。这些动力学障碍是如何编码的还不太清楚。在这里，我们使用 α-溶菌酶（αLP）蛋白家族中的进化序列变异来探测序列与能量景观之间的关系。αLP 具有不寻常的能量景观：αLP 的天然状态不是热力学上最有利的构象，而是由于阻止展开的大动力学障碍而保持折叠。为了折叠，αLP 利用与蛋白酶本身大小相似的 N 端前导区作为折叠催化剂。一旦折叠，前导区就被去除，天然状态不会在生物学相关的时间尺度上展开。没有前导区，αLP 的折叠时间约为千年。系统发育搜索揭示了具有广泛前导区大小的 αLP 同源物，包括一些完全没有前导区的同源物。在由此产生的系统发育树中，这些同源物按前导区大小聚类。通过研究天然缺乏前导区的同源物，我们证明它们可以在热力学上稳定，折叠速度比 αLP 快得多，但仍保留与 αLP 相同的折叠。被认为有助于 αLP 极端动力学稳定性的关键氨基酸在这些同源物中丢失，支持它们在动力学稳定性中的作用。这项研究强调了整个能量景观如何在决定蛋白质序列的进化压力方面发挥重要作用。
