Department of Chemical Engineering, Columbia University, New York, New York 10027, United States.
ACS Synth Biol. 2024 Aug 16;13(8):2600-2610. doi: 10.1021/acssynbio.4c00431. Epub 2024 Aug 7.
The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.
酶活性的工程设计通常涉及对蛋白质一级序列的改变，这些改变引入了结构变化，从而产生了功能的改善。机械力已被用于探究蛋白质生物物理学，从而在单分子研究中产生了深刻的机械见解。在这里，我们使用简单的 DNA 弹簧施加较小的拉力来扰动热稳定醇脱氢酶的活性位点。开发了方法来使不同的弹簧长度和弹簧方向在大规模催化条件下进行研究。施加在活性位点上的张力扩大了结合口袋的体积，并改变了酶对长链长度底物的偏好，这可以通过改变弹簧长度和产生的作用力来调节。当 DNA 弹簧被切断或旋转约 90°时，不会发生底物特异性的变化。这些发现展示了一种蛋白质工程的替代方法，其中可以使用施加的机械力动态且可逆地重塑活性位点结构。
