Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, Champaign, IL 61801, USA.
Phys Chem Chem Phys. 2019 Nov 13;21(44):24393-24405. doi: 10.1039/c8cp07775d.
Engineered repeat proteins have proven to be a fertile ground for studying the competition between folding, misfolding and transient aggregation of tethered protein domains. We examine the interplay between folding and inter-domain interactions of engineered FiP35 WW domain repeat proteins with n = 1 through 5 repeats. We characterize protein expression, thermal and guanidium melts, as well as laser T-jump kinetics. All experimental data is fitted by a global fitting model with two states per domain (U, N), plus a third state M to account for non-native states due to domain interactions present in all but the monomer. A detailed structural model is provided by coarse-grained simulated annealing using the AWSEM Hamiltonian. Tethered FiP35 WW domains with n = 2 and 3 domains are just slightly less stable than the monomer. The n = 4 oligomer is yet less stable, its expression yield is much lower than the monomer's, and depends on the purification tag used. The n = 5 plasmid did not express at all, indicating the sudden onset of aggregation past n = 4. Thus, tethered FiP35 has a critical nucleus size for inter-domain aggregation of n ≈ 4. According to our simulations, misfolded structures become increasingly prevalent as one proceeds from monomer to pentamer, with extended inter-domain beta sheets appearing first, then multi-sheet 'intramolecular amyloid' structures, and finally novel motifs containing alpha helices. We discuss the implications of our results for oligomeric aggregate formation and structure, transient aggregation of proteins whilst folding, as well as for protein evolution that starts with repeat proteins.
工程化重复蛋白已被证明是研究连接蛋白结构域折叠、错误折叠和瞬时聚集之间竞争的肥沃土壤。我们研究了工程化 FiP35 WW 结构域重复蛋白的 n = 1 至 5 个重复与折叠和结构域间相互作用之间的相互作用。我们对蛋白质表达、热和胍盐溶解以及激光 T-跳跃动力学进行了表征。所有实验数据均通过具有两个结构域（U、N）状态的全局拟合模型进行拟合，外加一个第三个状态 M，以解释由于所有非单体中存在的结构域相互作用而导致的非天然状态。使用 AWSEM 哈密顿量通过粗粒化模拟退火提供了详细的结构模型。具有 n = 2 和 3 个结构域的连接 FiP35 WW 结构域仅略低于单体的稳定性。n = 4 低聚物的稳定性更低，其表达产量远低于单体，并且取决于所使用的纯化标签。n = 5 质粒根本没有表达，表明在 n = 4 之后突然开始聚集。因此，连接 FiP35 具有 n ≈ 4 的结构域间聚集的关键核大小。根据我们的模拟，从单体到五聚体，错误折叠结构变得越来越普遍，首先出现扩展的结构域间β片层，然后出现多片层“分子内淀粉样”结构，最后出现含有α螺旋的新型基序。我们讨论了我们的结果对寡聚体聚集形成和结构、蛋白质折叠时的瞬时聚集以及从重复蛋白开始的蛋白质进化的影响。
