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

基于模拟，越来越多的理论研究声称存在不同的振动能量转移（VET）途径，这些途径连接蛋白质中重要的功能位点。分子内信息传递（变构）的影响进行了讨论。然而，这些高度定向的途径缺乏实验证据。需要开发适当的实验工具，以绘制出蛋白质中的VET，并仔细检查所提出的高度定向的途径的存在。在这里，我们提出通过将振动能以位点选择性注入蛋白质并随后实时跟踪其传播来对蛋白质中的VET途径进行直接实验研究-这是上述计算方法的实验对应部分。在蛋白质中绘制VET需要能够选择性地注入振动能，选择性地监测其流动，重要的是，能够灵活地选择相应的注入和探测位点。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。该对由VET供体和VET传感器组成，VET供体由飞秒泵浦脉冲激发，VET传感器的响应由飞秒红外探测脉冲测量。VET对的功能性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网站上和建议的传输路径，这将使我们能够比较实验的能量传输效率和时间尺度与理论预测的。将获得蛋白质中振动能量转移的详细图。通过这种方式，可以满足由变构位点之间的不同VET途径的理论预测所带来的实验挑战。我们希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。

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.