Site-Specific¹⁹F Incorporation For Protein Structure And Dynamics Studies Using¹⁹F Nuclear Magnetic Resonance

In this thesis, we will discuss the method of combining unnatural amino acid labeling and nuclear magnetic resonance to study protein structure and dynamics. There are six chapters in this thesis.Chapter1is a review of protein structure, function and dynamics, as well as the methods of nuclear magnetic resonance in the study of protein structure, function and dynamics. Protein is the basis of life activities. The function of protein is inextricably linked with stuctures. The structure of proteins is the basis of protein function and activity. Protein structure is intrinsically dynamic and continually performed conformational changes on a wide range of timescales. Protein dynamics affect a wide range of functions, such as ligand binding, folding and thermostability. Dynamics can enable some proteins to perform multiple different functions. Dynamics are important for the evolution of novel functions of proteins. NMR spectroscopy is uniquely suited to study protein dynamics. The most fundamental limitation of NMR is the poor sensitivity of signal detection. This places NMR a relatively low upper-limit on the size of proteins which can be studied. Spectral crowding is also a problem to NMR. However, those limitations can be solved by solid state NMR and site-specific amino acid labeling at some degree.Chapter2is a review of site-specific labeling of unnatural amino acid to study protein structure and dynamics. NMR is currently capable of studying the structure, ligand interactions and conformational changes of proteins and complexes approaching a megadalton in size. The ability to extend NMR to such large systems has been affected by improvements in instrumentation, experiments and isotope labeling approaches for large proteins. However, the increased number of NMR correlations and broad linewidth associated with larger proteins will generally result in more congested spectra. Then there will be dufficult to assign. A simple approach to reduce signal complexity is using amino acid type labeling methods. Unfortunately these methods are limited when applied to larger systems. Because assigning a large number of identical amino acids still a challenge. Site-specific labeling with unnatural amino acids represents a powerful method because assignment is straightforward. We will introduce kinds of NMR-active unnatural amino acids and methods of labeling unnatural amino acids incorporated into proteins. Further more liquid and solid state NMR in the study of non-natural amino acids will be introduced.In chapter3, the compound15N/19F tfmF was synthetized successfully. An orthogonal amber tRNA/tRNA synthetase pair for15N/19F-trifluoromethyl-Phenyl-alanine (15N/19F-tfmF) has been applied to achieve site-specific labeling of SH3at three different sites. One dimensional solution NMR spectra of backbone amide (15N)1H and side-chain19F were obtained for SH3with three different site-specific labels. Site-specific backbone amide (15N)1H and side-chain19F chemical shift and relaxation analysis of SH3in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. One dimensional19F spectra and T1, T2relaxation data were acquired on a SH3domain in aqueous buffer containing60%glycerol, and a nine-transmembrane helices membrane protein diacyl-glycerol kinase (DAGK) in dodecyl phosphochoine (DPC) micelles. Site-specific19F chemical shift and side chain relaxation analysis can be applied to site-specifically analyze side chain internal mobility of membrane proteins or large size proteins.In chapter4, the more application of site-specific F NMR will be discussed. Firstly,19F-tfmF was applied to accomplish site-specific19F spin incorporation at different sites in diacylglycerol kinase (DAGK, an Escherichia coli membrane protein) for site-specific solvent exposure analysis. Due to isotope effect on19F spins, a standard curve for19F-tfmF chemical shifts was drawn for varying solvent H2O/D2O ratios. The acquired solvent isotope shift values for the seven DAGK/DPC sites were consistent with the residue distribution in a trimeric membrane protein with three transmembrane helices in each monomer. Secondly,19F was site-specifically introduced to L27tan via the incorporation of tfmF. Different19F signal intensity attenuations were observed at different L27tan sites. It is due to different distances between the site-specifically incorporated tfmF and site-directed spin radical labeling. Analysis of the19F detection PRE showed that the L27tan protein had a closed conformation in solution. Thirdly, Site-specific F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK) were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that19F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while19F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.In chapter5, solution NMR characterization of SO-S1loop of BK channel a subunit will be discussed. BK channel is a large conductance potassium channel, which could be activated by intracellular Ca+, Mg2+as well as by membrane depolarization, and plays a central role in numerous physiological processes. The SO-Sl loop of BK channel is essential to its structure and function. Multidimensional heteronuclear nuclear magnetic resonance spectroscopy was used to study the secondary structure and its backbone dynamics of the S0-S1loop of BK channel. There are two amphiphilic helices in the S0-S1loop which might interact with the membrane. Two possible Mg2+binding sites in the S0-S1loop were studied by Mg2+titration experiments.Chapter6is the main expectations for how to combine protein structure and dynamics using the methods of19F un-natural amino acid labeling. The functional diversity of membrane proteins is determined not only by membrane proteins themselves, but also by their interactions with membranes. Native cellular membranes host various proteins, together with lipids in a variety of compositions. How to analysis membrane proteins’structure in its native membrane will be discussed.