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具有重要功能的部位。讨论了分子内信息传递(变构)的含义。然而，这些高度定向的通路缺乏实验证据。需要开发合适的实验工具，以便绘制出VET在蛋白质中的图谱，并仔细检查所提出的高度定向通路的存在。在这里，我们建议通过向具有位置选择性的蛋白质注入振动能量并随后跟踪其实时传播来直接实验研究蛋白质中的VET通路-这是上述计算方法的实验对应物。绘制VET在蛋白质中的图谱需要有能力选择性地注入振动能量，选择性地监测其流动，更重要的是，要有灵活性地选择各自的注射和探测位置。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(NCAA)来实现这一点。该对由一个由飞秒泵浦脉冲激发的VET供体和一个由飞秒红外探测脉冲测量的VET传感器组成。VET对的功能已经在我们关于小合成肽的初步工作中得到了证明。在这里，我们将把我们的前期工作从简单的多肽扩展到蛋白质。我们将使用扩展遗传密码的方法，以实现VET供体和感受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所提出的转移路径上和之外的不同位置，这将使我们能够将实验的能量转移效率和时间尺度与理论预测的结果进行比较。我们将获得蛋白质中振动能量转移的详细图谱。通过这种方式，可以应对变构中心之间不同的VET路径的理论预测所带来的实验挑战。我们期望能够在实验上证明(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.