Verifying the Concept of Extreme Electric Fields in Proteins using Experimentally Refined Vibrational Spectroscopic Maps

使用实验精制的振动光谱图验证蛋白质中极端电场的概念

• 批准号: 493270578
• 负责人: Dr. Jacek Artur Kozuch
• 金额: --
• 依托单位: Institut für Experimentalphysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/493270578?language=en
• 关键词: Verifying; Concept; Extreme; Electric; Fields

Vibrational spectroscopy is a versatile methodology that enables exploring various physical chemical phenomena, such as electrostatic contributions to (bio)chemical process. A simple framework to describe the vibrational-electrostatic response is the linear vibrational Stark effect (VSE), which uses vibrational probes (e.g., the C=O stretch) as electric field (EF) sensors at functionally relevant locations, such as in catalyst active sites. A recent milestone of VSE applications was the direct infrared (IR) spectroscopic evidence for extreme active site EFs along reactive C=O bonds in enzyme active sites, lowering the reaction’s activation barriers via the mechanism of electrostatic catalysis. For the model enzyme ketosteroid isomerase, vibrational peak shifts of -100 cm-1 were found, which are consistent with fields of -140 MV/cm using the linear VSE. Recently, we reported even higher shifts and EFs of -140 cm-1 and -175 MV/cm, respectively, for TEM β-lactamases, which are responsible for antibiotic resistances. These EFs present a highly relevant measure to understand (or even predict) the evolutionary fitness landscape to antibiotic resistance or to guide the design of de novo enzymes in biocatalysis. However, one criticism of the linear VSE is that it neglects many other contributions to vibrational peak shifts, that should be considered for such extreme shifts and EFs. These contributions can include polarizability, EF gradients, dispersion, Pauli repulsion, vibrational coupling, etc., and their neglect can lead to inaccurately determined EFs and to erroneous conclusions. The aim of this work is to systematically test the applicability of the linear VSE for cases of extreme peak shifts and EFs using a combined experimental and computational approach centered around vibrational spectroscopic maps (VSM) for the prediction of IR spectra from polarizable molecular dynamics (AMOEBA MD) simulations. VSMs model vibrational observables via semiempirical or high-level quantum mechanical parameterizations of their response to interactions with the local environment. In this work, VSM parameters compatible with high peak shifts and EFs will be generated for the ligands and the enzyme active site environment based on high-level density functional theory, and refined experimentally using vibrational Stark spectroscopy. In the latter, the VSE is detected in controlled, external electric fields enabling to directly determine electrostatic parameters as experimental constraints in the VSM parameterization towards physically accurate parameterizations. The VSM will be utilized to predict IR spectra from polarizable AMOEBA MD simulations of the enzyme/ligand systems in order to (re-)interpret experimental peak shifts. In this way, we will test the reliability of the linear VSE in predicting extreme EFs and refine our understanding of vibrational spectra in the peculiar environment of the enzyme active site.

振动光谱学是一种通用的方法，可以探索各种物理化学现象，如静电对（生物）化学过程的贡献。描述振动-静电响应的一个简单框架是线性振动斯塔克效应（VSE），它使用振动探针（例如C=O拉伸）作为在功能相关位置（例如催化剂活性位点）的电场（EF）传感器。最近VSE应用的一个里程碑是直接红外（IR）光谱证据，证明了酶活性位点上沿活性C=O键的极端活性位点EFs，通过静电催化机制降低了反应的激活障碍。对于模型酶酮类固醇异构酶，发现振动峰位移为-100 cm-1，与线性VSE的-140 MV/cm场一致。最近，我们报道了TEM β-内酰胺酶的更高位移和电场分别为-140 cm-1和-175 MV/cm，这是抗生素耐药的原因。这些EFs为理解（甚至预测）抗生素耐药性的进化适应性景观或指导生物催化中新生酶的设计提供了高度相关的措施。然而，对线性VSE的一个批评是，它忽略了许多其他对振动峰值位移的贡献，这些贡献应该被考虑到这种极端位移和电场。这些贡献包括极化率、EF梯度、色散、泡利斥力、振动耦合等，忽略它们可能导致不准确地确定电磁场并得出错误的结论。这项工作的目的是系统地测试线性VSE在极端峰移和EFs情况下的适用性，使用以振动光谱图（VSM）为中心的实验和计算相结合的方法来预测极化分子动力学（AMOEBA MD）模拟的红外光谱。VSMs通过半经验或高级量子力学参数化来模拟振动可观测物对其与局部环境相互作用的响应。本研究将基于高密度泛函理论，对配体和酶活性位点环境生成与高峰移和电场相兼容的VSM参数，并利用振动Stark光谱进行实验细化。在后者中，VSE是在受控的外部电场中检测的，从而可以直接确定静电参数，作为VSM参数化的实验约束，以实现物理精确的参数化。VSM将用于预测酶/配体系统的极化AMOEBA MD模拟的红外光谱，以便（重新）解释实验峰移。通过这种方式，我们将测试线性VSE在预测极端电场方面的可靠性，并完善我们对酶活性位点特殊环境下振动谱的理解。