In-situ Protein-protein Interaction And Function Analysis Using Site-Specific¹⁹F Incorporation And¹⁹F Nuclear Magnetic Resonance

In this dissertation, we describe the basis of methods for unnatural amino acids incorporation and their diverse applications in different areas. We also discuss specific advantages of the fluorine nucleus for NMR investigations and their diverse applications. Combination of unnatural amino acids incorporation and19F NMR will be a powerful tool to study protein structure and function. There are six chapters in this thesis.A review of methods for unnatural amino acids incorporation and their diverse applications is introduced in chapter1. The ability to incorporate unnatural amino acids into proteins at specific sites has begun to have a direct impact on biological processes study that are difficult to address by other classical methods. One of the most powerful ways for incorporating unnatural amino acids site-specifically into proteins is genetic code expansion. In this approach, an aminoacyl-tRNA synthetase/tRNA pair is used to specifically insert the unnatural amino acid in response to an amber stop codon (TAG), which is placed at a defined site in a gene of interest. Native chemical ligation will be a complementary way for site-specific incorporation. Unnatural amino acids can also be incorporated residue-specifically throughout the proteome by an approach known as selective pressure incorporation. However, this will lead to an overall incorporation of unnatural amino acids, rather than site-specific incorporation by genetic code expansion. At the end of this chapter, we focus on diverse applications of unnatural amino acids incorporation in different areas, including in some new frontiers.In chapter2, we apply19F NMR to study protein structure and function.19F NMR is another effective approach for protein conformation or function studies, especially with the100%natural abundance and high gyromagnetic ratio of19F spins. With the low or none background natural abundance interference from biological samples, high sensitivity of19F chemical shift to protein conformational changes or ligand binding and few structural perturbations for19F incorporation into proteins. In the first section of this chapter, we summarize the specific advantages of the fluorine nucleus for NMR investigations, then we discuss methods available to prepare fluorine-labeled proteins and peptides. In the end, we mainly focus on the application of19F NMR in combination with fluorinated molecular probes in diverse areas, including structural and functional studies of proteins and nucleic acids, drug screening and discovery, metabolism analysis and in vivo tracking of bioactive molecules.In chapter3, we apply19F NMR in combination with site-specific unnatural amino acids incorporation to analysis protein-protein interaction in bacterial crude lysate. We successfully synthesised unnatural amino acids trifluoromethyl phenylalanine. The protein-protein interaction analysis in native cellular environment using19F NMR was exemplified with two PB1domains from two mitogen-activated protein kinases (MAPKs):MEKK3or MEK5. MAPK superfamily is one of the major systems participating in the transduction of signals from the cell membrane to nuclear and other intracellular targets, playing a crucial role in cell function. Both MEKK3and MEK5harbor a PB1domain in the N-terminus, and they form a heterodimer via PB1-PB1domain interaction. The19F-tfmF was incorporated into different sites of MEKK3-PB1or MEK5-PB1respectively. Then,19F NMR chemical shift and relaxation were measured to analyze interaction interfaces between MEKK3-PB1and MEK5-PB1. Residue sites with chemical shift perturbations in the interfacial region of MEKK3-PB1/MEK5-PB1after protein co-purification were demonstrated to be consistent with the chemical shift perturbation analysis of the protein complex in crude bacterial cell lysate.In chapter4, we discuss nano-size uni-lamellar lipodisq improved in situ auto-phosphorylation analysis of E. coli tyrosine kinase using19F nuclear magnetic resonance. In this chapter, we presented the advantage of correlational structure and function studies of membrane proteins in nano-size uni-lamellar lipodisq with SMA surrounding the lipid bilayer, even native cell membrane. Other alternative nano-size unilamellar membrane configurations, such as bicelles and nanodiscs, were reported to improve substrate accessibilities to two sides of an incorporated membrane protein in lipid bilayers. However, specific lipid, detergents are required or membrane scaffold protein (MSP) are required. Besides the well integrity maintenance of the native environment and the integral membrane proteins in uni-lamellar lipodisq, the improved accessibility of soluble partner to SMA based lipodisqs in homogenous size provided much convenience for further receptor ligand binding, enzymology analysis and protein-protein interaction analysis in native cell membrane environment using19F solid state NMR or other biophysical methods.In chapter5, we discuss the history of small molecule fluorescent probes and their applications in different areas. We combine unnatural amino acids incorporation and photophysical properties of small molecule fluorescent probe L-(7-hydroxycoumarin- 4-yl) ethylglycine to study protein structure and function.In chapter6, a brief review of the future of unnatural amino acids incorporation and19F NMR is discussed. It is important to study protein structure and function in native membrane environment, which will reflect the real physiological properties. Rapid development of unnatural amino acids incorporation and19F NMR will be a powerful tool to analysis biophysical information.