Abstract:
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.
摘要:
酶活性的工程通常涉及蛋白质一级序列的改变,引入结构变化,从而导致功能改进。机械力已经被用来询问蛋白质生物物理学,导致在单分子研究中深入的机械见解。这里,我们使用简单的DNA弹簧施加小的拉力来干扰热稳定的醇脱氢酶的活性位点。开发了一些方法来研究本体催化条件下的不同弹簧长度和弹簧取向。跨活性位点施加的张力扩大了结合袋体积,并改变了酶对更长链长底物的偏好,这可以通过改变弹簧长度和所施加的力进行调整。当DNA弹簧被切断或旋转~90°时,底物特异性变化不会发生。这些发现证明了蛋白质工程中的另一种方法,其中活动站点架构可以使用施加的机械力动态和可逆地改造。