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.
