The interplay between specificity and stability in lactamases: molecular modeling of flexibility and dynamics

在拟议的项目中，我们开发了一个通用的分子模型来描述和预测丝氨酸内酰胺酶的结构、灵活性和动力学是如何通过不稳定的突变来修改的，这些突变提高了特异性，从而转移了广谱内酰胺酶的活性或抑制剂耐药性，以及增加了稳定性但对活性没有影响的突变。我们的工作假设是，失稳和稳定可以由两种机制中的任何一种产生：一种是众所周知的局部效应，如堆积、静电、氢键和溶剂效应，另一种是长期效应，如最近才提出的主干柔韧性增加和运动耦合。我们将通过大量的多分子动力学模拟和从头开始的蛋白质设计工具来研究这些机制。了解不稳定突变和稳定突变的相互作用将有助于理解自然进化，并提供一种可普遍用于蛋白质工程的设计策略。我们模型的预测通过与三个实验伙伴的合作得到验证：(1)通过定向进化和工程突变的生化特征检测新的稳定突变(在SPP 1170框架下，Freiburg的Kristian Müler教授)。(2)如果前景看好，将进行蛋白质骨架灵活性的核磁共振研究(南安普顿大学的Jörn Werner博士)(3)从临床样本中分离出的内酰胺酶变体的分析(PD，斯图加特工业生化研究所的Till Bachmann博士)。

In the proposed project we develop a general molecular model to describe and predict how structure, flexibility, and dynamics of serine lactamases is modified by destabilizing mutations that improve specificity, thus transferring extended spectrum lactamase activity or inhibitor resistance, and mutations that increase stability, but have no effect to activity. Our working hypothesis is that destabilization and stabilization could result from either of two mechanism: from well-known local effects like packing, electrostatics, hydrogen bonding, and solvent effects, or from long-range effects like increased backbone flexibility and coupling of motions, as it has been suggested only recently. We will study these mechanisms by massive multiple molecular dynamics simulations and de novo protein design tools. Understanding the interplay of destabilizing and stabilizing mutations would help to understand natural evolution and provide a design strategy which could be generally used in protein engineering. Predictions of our model are validated by a co-operation with three experimental partners: (1) Detection of new stabilizing mutants by directed evolution and biochemical characterization of engineered mutants (Prof. Dr. Kristian Müller, Freiburg, in the framework of SPP 1170). (2) If promising, an NMR study of protein backbone flexibility will be carried out (Dr. Jörn Werner, University of Southampton) (3) Analysis of lactamase variants isolated from clinical samples (PD Dr. Till Bachmann, Institute of Technical Biochemistry, Stuttgart).