The Studies Of Protein Labeling Strategies In Magic Angle Spinning Solid-state NMR

Magic Angle Spinning Solid-State NMR (MAS SSNMR) has its unique advantages to reveal the atomic-level structure and dynamics of proteins which are insoluble in water or difficult to crystallize, such as membrane proteins. As one class of important proteins, membrane proteins play a critical role in material transportation, cell signaling and energy transformation. About 70% of the drugs on the market are designed to target membrane proteins. Hence, the determination of membrane proteins structures will accelerate greatly the design of medicines to improve human health. However, membrane proteins structure determination is very challenging due to its general properties of low expression, hydrophobicity and instability. MAS SSNMR can study structure and dynamics of membrane proteins directly in lipid bilayers, which are closer to the natural environment. In addition, MAS SSNMR can determine infinitely large-size protein structures in theory. Consequently, MAS SSNMR is expected to be a potentially technique to elucidate the structure and dynamics of membrane proteins.Nevertheless, difficulties in obtaining sufficient structure restraints hamper the progress of MAS SSNMR in protein structure determination. It mainly dues to three reasons. First, It’s difficult to obtain distance restraints assignment from seriously overlapping spectrum in MAS SSNMR. Second,13C and 15N nucleus are the main directly detected nucleus in MAS SSNMR. Both of them have very low sensitivity. As a result, it is extremely difficult to obtain distance restraints by 3D or 4D MAS SSNMR experiments. The third reason, the difficulties in obtaining long-range distance restraints (>6 A) by traditional methods arise from the weak dipole-dipole couplings between 1H,13C, and 15N, which are proportional to nuclear gyro magnetic ratios of those spins and also to the inverse third power of the nucleus-nucleus distance. However, restraints corresponding to long distance (>6 A) are crucially important in defining the global protein folding, especially for multiple domains or large-size proteins.To overcome these limitations requires development of new techniques, such as labeling techniques. Consequently, this doctoral dissertation mainly focused on developing various labeling methods including different isotope labeling strategies and paramagnetic labeling techniques in MAS SSNMR. By utilizing these labeling methods, we studied the homotrimer interface and 3D structure of membrane protein diacylgycerol kinase (DAGK) in E.coli membrane extracts.It primarily discussed the selective unlabeled efficiency of different amino acids in model protein of GB1. Seven kinds of amino acids, including Phe, Trp, Tyr, Ile, Leu, Lys and Thr, could be simultaneously unlabeled when [U-13C]-Gluclose was used as the sole carbon source to expressed GB1. We compared the alternatively labeling efficiency on membrane protein DAGK using [1,3-13C]-Glycerol or [2-13C]-Glycerol as the sole carbon source with the ones using [1-13C]-Glucose or [2-13C]-Glucose as sole carbon source. DAGK alternatively labeled by [1,3-13C]-Glycerol or [2-13C]-Glycerol had a little better resolution and much higher signal-to-noise ratio spectrum than those labeled by [1-13C]-Glucose or [2-13C]-Glucose. It proved that carbon source of [1,3-13C]-Glycerol or [2-13C]-Glycerol were more suitable to alternatively label membrane protein for structure studying.In the second work, we have shown that high-resolution structure of diamagnetic proteins in solid phase could be generated from a combination of MAS SSNMR PCS measurements and Rosetta calculations.2D high-resolution solid-state MAS SSNMR spectra of U-13C,15N enriched model protein GB1 containing covalently paramagnetic tags provided long-range structural restraints of 10-20 A which were inaccessible to a dipolar coupling based approach. We also have shown the flexibility of introduction of different paramagnetic ions to different sites of the protein, enabling the coverage of several fragments of the protein and yielding more complete PCS-derived distance maps. This work indicated that using PCSs as a source of long-range restraints could be a general route to structure determination of challenging biomacromolecules, such as membrane proteins and amyloid fibrils by solid-state NMR.In the third work, we studied the homotrimer interface and 3D structure of membrane protein DAGK in E. coli membrane extracts by combining various isotopic labeling methods with paramagnetic labeling technique. In this work, we found the way to make DAGK unfold to monomer and then DAGK monomer refold back to trimer. Not only DAGK would maintain its native struture, but also the enzyme still had good activity after unfolding and refolding. Based on the freely transformation between trimer and monomer of DAGK, it’s doable to perform TEDOR, PDSD and intermolecular paramagnetic relaxation enhancement (PRE) experiments to probe the homotrimer interface of DAGK. According to 31 intermolecular distance restraints obtained from intermolecular PREs, the overall quaternary structure of DAGK in E. coli membrane extracts determinated by MAS SSNMR was consistent with both revealed in DPC detergent by Solution NMR and in lipd cubic phase (LCP) X-ray crystallography. However, there were still notable disparities. Moreover, It’s the first time to exploit TEDOR, PDSD and intermolecular PREs based on a mixing sample of paramagnetic labeling and isotope labeling to detect quaternary structure of membrane proteins. After comparison and contrast, intermolecular PREs, which is easier to perform NMR experiments and assign much longer and more abundant distance restraints, had more advantages to probe oligomerization interface of a membrane protein than TEDOR and PDSD. It indicated intermolecular PREs could be a routine method to reveal quaternary structure of membrane proteins. Meanwhile, we combined distance restraints from intra-molecular PRE with those from traditional dipolar-dipolar coupling based DARR and PDSD to elucidate 3D structure of DAGK reconstituted in E. coli membrane extracts.