Mehlich Alexander, Fang Jie, Pelz Benjamin, Li Hongbin, Stigler Johannes
Physics Department E22, Technische Universität München, Garching, Germany.
Department of Chemistry, University of British Columbia, Vancouver, BC, Canada.
Front Chem. 2020 Dec 7;8:587824. doi: 10.3389/fchem.2020.587824. eCollection 2020.
designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins.
设计蛋白作为纳米工程和生物医学的潜在平台受到了广泛关注。虽然在设计热力学稳定蛋白方面已经开展了大量工作，但人工设计蛋白的折叠过程尚未得到充分研究。在此，我们利用光镊单分子力谱技术研究了一种设计的2x2罗斯曼折叠结构ROSS的折叠过程。我们测量到的势垒穿越时间在毫秒范围内，比其他系统报道的要慢得多。虽然长过渡时间可以用势垒粗糙度或缓慢扩散来解释，但我们表明各向同性粗糙度无法解释测得的过渡路径时间分布。相反，这项研究表明ROSS的缓慢势垒穿越是由三种短寿命高能中间体的存在引起的。此外，我们识别出具有不同势垒穿越动力学的不完全和偏离路径的折叠事件。我们的结果暗示存在一个复杂的过渡势垒，这可能是许多人工设计蛋白的共同特征。
