Quantifying the contribution of a new type of H-bond to protein stability

蛋白质是氨基酸的线性链，通过涉及其主链（即主链）原子以及不同的单个氨基酸侧链原子的各种分子内相互作用，折叠成复杂的三维结构。在蛋白质中提供结构稳定性的一种非常有特征的相互作用是氢键（h -键），这是一种静电相互作用，形成于具有相反极性的蛋白质主原子之间，并以结构上可接近的方式定位。该项目研究了蛋白质中另一种以前未被充分认识的稳定相互作用的作用，即顺序相邻氨基酸的主链原子之间的氢键。该项目将使用一种实验方法，依靠碳-氘（C-D）键来可视化特定的蛋白质振动，以研究这种类型的氢键对蛋白质稳定性的能量贡献。定义蛋白质折叠、结构和稳定性的相互作用都是活性和功能的重要因素，因此识别和量化新型相互作用的研究有可能影响蛋白质科学各个方面的基础研究。该项目将为研究生、本科生和高中生提供教育和培训，让他们参与项目的各个方面。N-H给体和CO受体之间形成的氢键一直被认为是蛋白质结构和稳定性的主导力量。然而，蛋白质内的许多残基的构象分别为φ和psi值约为±90°和0°，传统上认为这是由于酰胺态氮（Ni）和随后的氨基酸（Ni+1）之间的空间冲突而被禁止的。该项目将研究这些构象中形成的(Ni+1)-H- (Ni)相互作用，这些构象构成了以前未被重视的氢键类型。该项目的目标是明确确定R67 DHFR中(Ni+1)-H—（Ni）氢键介导的稳定性。该项目将比较其(Ni+)-H—（Ni）氢键完整的蛋白质与等构CH2类似物的稳定性[其中一个或多个(Ni+1)-H—（Ni）氢键被破坏]。此外，模型系统将用于生成校准曲线，该曲线将能够解释在稳定方面观察到的蛋白质红外光谱的变化。目标的成功完成将提供一个令人信服的证明（Ni+1-H）—（Ni）氢键的形成，重要的是，将提供它们对蛋白质稳定性贡献的衡量标准。

Proteins are linear chains of amino acids that fold into complex three-dimensional structures by virtue of a variety of intramolecular interactions involving both their main chain (i.e. backbone) atoms, as well as the different, individual amino acid side chain atoms. One very well characterized interaction in proteins that provides structural stability is the hydrogen bond (H-bond), which is a type of electrostatic interaction formed between protein backbone atoms that have opposite polarity and positioned in a structurally accessible manner. This project investigates the role of an additional and previously underappreciated type of stabilizing interaction in proteins, H-bonds between backbone atoms of sequentially adjacent amino acids. This project will use an experimental approach that relies on the use of carbon-deuterium (C-D) bonds to visualize specific protein vibrations to investigate energetic contribution of this type of H-bond in protein stability. The interactions that define a protein's folding, structure, and stability are all important factors in activity and function, thus research to identify and quantify novel types of interactions have the potential to impact basic research into all aspects of protein science. This project will provide education and training for graduate, undergraduate and high school students by involving them in every aspect of the project. H-bonds formed between backbone N-H donor and CO acceptors have long been appreciated as dominant forces underlying protein structure and stability. However, many residues within proteins adopt conformations with phi and psi values of approximately ±90° and 0°, respectively, which are traditionally considered forbidden due to a steric clash between the amide nitrogen (Ni) and that of the following amino acid (Ni+1). This project will investigate the (Ni+1)-H---(Ni) interactions formed within these conformations which constitute a previously underappreciated type of H-bond. The objectives of this project is to focus specifically on determining the stabilization mediated by the (Ni+1)-H---(Ni) H-bonds in R67 DHFR. This project will compare the stability of the protein with its (Ni+)-H---(Ni) H-bonds intact with that of an isosteric CH2 analog [wherein one or more of th(Ni+1)-H---(Ni) H-bonds are disrupted]. In addition, model systems will be used to generate a calibration curve that will enable an interpretation of the shifts in IR spectra observed with the protein in terms of stabilization. Successful completion of the objectives will provide a convincing demonstration that (Ni+1-H)---(Ni) H-bonds are formed, and importantly, will provide a gauge of their contribution to protein stability.