School of Molecular Sciences, Arizona State University, Tempe, AZ 85281, USA.
The Biodesign Center for Molecular Design and Biomimetics, Arizona State University, Tempe, AZ 85281, USA.
Int J Mol Sci. 2021 Mar 12;22(6):2895. doi: 10.3390/ijms22062895.
The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.
蛋白质运动（即动力学）与酶功能之间的关系已开始在β-内酰胺酶中进行探索，以期深入了解这些蛋白质。在最近的一项研究中，我们分析了 TEM-1（一种普遍存在的 A 类β-内酰胺酶）和几个祖先重建的同源物的动态特征。这项工作的主要发现是，与活性位点别构偶联的刚性残基似乎对酶功能有深远影响，即使它们与活性位点相隔数埃。在目前的工作中，我们的目的是通过使用计算蛋白质设计方法改变 TEM-1 的动态特征，进一步探讨蛋白质动力学对β-内酰胺酶功能的影响。Rosetta 软件套件用于突变与活性位点高度偶联的刚性残基周围的氨基酸，或与活性位点没有明显通信的柔性残基周围的氨基酸。对十个设计蛋白质的实验表征表明，改变与刚性、高度偶联残基周围的残基，会显著影响酶的活性和稳定性；相比之下，当靶向柔性、不偶联的残基时，保持了类似天然的活性和稳定性。我们的结果为 TEM 家族β-内酰胺酶中存在的结构-功能关系提供了更多的见解。此外，计算蛋白质设计方法与蛋白质动力学分析的结合代表了一种通用方法，可以用于扩展我们对其他酶类中动力学与功能之间关系的理解。
