Mapping vibrational energy transfer in proteins and investigating its relation to allosteric signal propagation using a non-canonical VET pair

使用非规范 VET 对绘制蛋白质中的振动能量转移图并研究其与变构信号传播的关系

• 批准号: 349128007
• 负责人: Professor Dr. Jens Bredenbeck
• 金额: --
• 依托单位: Institut für Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/349128007?language=en
• 关键词: Mapping; vibrational; energy; transfer; proteins

Based on simulations a growing number of theoretical studies claim the existence of distinct vibrational energy transfer (VET) pathways, which connect sites of functional importance in proteins. Implications for intramolecular information transfer (allostery) have been discussed. However, experimental evidence for these highly directional pathways is lacking. The appropriate experimental tools need to be developed in order to map out VET in proteins and to scrutinize the existence of the proposed highly directional pathways. Here, we propose a direct experimental investigation of VET pathways in proteins by injecting vibrational energy into a protein with site selectivity and subsequently following its propagation in real-time - which is the experimental counter-part of above mentioned computational approaches. Mapping out VET in proteins requires the ability to site-selectively inject vibrational energy, site-selectively monitor its flow and, importantly, to have the flexibility to chose the respective sites for injection and probing. We aim to achieve this by using our recently developed VET pair of non-canonical amino acids (ncAAs). The pair consists of a VET donor, which is excited by a femtosecond pump pulse and a VET sensor, whose response is measured by a femtosecond infrared probe pulse. The functionality of the VET pair has been demonstrated in our preliminary work on small synthetic peptides. Here, we will expand our preliminary work from simple peptides to proteins. We will use methods for expanding the genetic code in order to achieve precise positioning of VET donor and sensor in the target protein sequences and to obtain the large number and quantities of the required protein mutants by in vivo expression. Donor and sensor will be positioned at various sites both on and off the proposed transfer pathways, which will enable us to compare the experimental energy transfer efficiencies and time scales with the theoretically predicted ones. Detailed maps of vibrational energy transfer in proteins will be obtained. In this way, the experimental challenge posed by the theoretical prediction of distinct VET pathways between allosteric sites can be met. We expect to be able to experimentally proof (or reject) the existence of highly directional VET pathways and their relevance in allosteric signal transmission.

基于模拟，越来越多的理论研究声称存在不同的振动能量传递（VET）途径，这些途径连接了蛋白质中重要功能的位点。讨论了分子内信息传递（变构）的意义。然而，缺乏这些高度定向通路的实验证据。需要开发适当的实验工具，以便绘制蛋白质中的VET，并仔细检查所提出的高度定向途径的存在。在这里，我们提出了一种直接的实验研究，通过将振动能量注入具有位点选择性的蛋白质，随后实时跟踪其繁殖，这是上述计算方法的实验对应部分。绘制蛋白质中的VET需要有选择性地注入振动能量的能力，有选择性地监测其流动，重要的是，要有灵活性来选择注射和探测的相应位置。我们的目标是通过使用我们最近开发的VET对非规范氨基酸（ncAAs）来实现这一目标。这一对由一个VET供体和一个VET传感器组成，前者由飞秒泵脉冲激发，后者的响应由飞秒红外探针脉冲测量。VET对的功能已经在我们对小合成肽的初步工作中得到证实。在这里，我们将把我们的初步工作从简单的肽扩展到蛋白质。我们将使用扩展遗传密码的方法，以实现VET供体和传感器在靶蛋白序列中的精确定位，并通过体内表达获得大量所需的蛋白突变体。供体和传感器将被放置在不同的位置上，无论是在提出的转移途径，这将使我们能够比较实验的能量转移效率和时间尺度与理论预测的。将获得蛋白质中振动能量传递的详细图。通过这种方式，可以满足理论预测变构位点之间不同VET途径所带来的实验挑战。我们期望能够通过实验证明（或拒绝）高度定向的VET通路的存在及其在变构信号传递中的相关性。