ProtonsInProteins: A novel approach for studying biological proton transfer

蛋白质内部的质子转移（PT）反应是生物系统的基础，例如ATP的产生。到目前为止，还没有直接的方法来测量蛋白质中特定的PT途径，大多数研究都是基于通过天然质子途径跟踪PT反应的最终产物。在这里，我们的目标是开发一种直接测量蛋白质内PT的新方法。我们的新方法是基于将质子供体和受体放置在蛋白质的特定位置，其中PT只有在系统的光激发后才会启动。为此，我们在这里介绍了我们开发的两种非规范氨基酸（ncAA），光酸和光碱作为它们的残基分别作为质子供体和受体。我们的假设是，激发态PT的强光触发驱动力（约11 pKa单位）将沿着从供体到受体的途径启动PT。在第一个目标中，我们将使用我们新的ncAA与固相肽合成来设计几个肽系统，这将使我们能够破译特定氨基酸的作用，肽结构，以及水在肽中的作用，使用各种超快光谱。在第二个目标中，我们的目标是设计一个相互正交的系统，将我们的两个ncAA插入到单个蛋白质中。在我们的第三个目标中，我们计划使用我们的新实验系统来回答生物PT领域以前无法回答的一系列独特问题，重点关注ATP合酶跨膜复合体和可溶性碳酸酐酶系统。我们的新方法在生物学研究和理解PT方面具有开创性，并将使研究人员拥有全新的能力，从而产生令人着迷的新发现。此外，我们的新系统可以翻译到其他需要蛋白质内质子浓度局部变化的生物学领域。

Proton transfer (PT) reactions within proteins are fundamental in biological systems, such as within ATP production. To date, there are no direct means to measure specific PT pathways within proteins, and most research is based on following the end-product of the PT reaction across a natural proton pathway. Here, our goal is to develop a novel way to directly measure PT within proteins. Our new approach is based on placing proton donors and acceptors in specific places within proteins, in which the PT will be initiated only after light excitation of the system. To do so, we introduce here two noncanonical amino acids (ncAA) we developed, with a photoacid and a photobase as their residues that serve as the proton donor and acceptor, respectively. Our hypothesis is that the strong light-triggered driving force of PT in the excited-state (of ~11 pKa units) will initiate PT along the pathway from donor to acceptor. In the first objective, we will use our new ncAA with solid-phase peptide synthesis to design several peptide systems that will allow us to decipher the role of specific amino acids, the peptide structure, and the role of water in PT across the peptide using various ultrafast spectroscopy. In the second objective, we aim to design a mutually orthogonal system for the insertion of our two ncAA into a single protein. In our third objective, we plan to use our new experimental system for answering a unique set of questions in the field of biological PT that could not have been answered before, focusing on the systems of ATP synthase transmembrane complex and the soluble carbonic anhydrase enzyme. Our new approach is groundbreaking in the way we study and understand PT in biology and will enable researchers completely new capabilities resulting in fascinating new discoveries. Moreover, our new system can be translated into other fields in biology that require the local change in proton concentration within proteins.