Fast Four-dimensional NMR Experiments For Backbone Chemical Shift Assignment Of Intrinsically Disordered Proteins And Methyl-methyl NOESY Experiment

After years of development, NMR spectroscopy has become one of the most important tools for exploring the structural and dynamic characteristics of macromolecules, such as proteins and nucleic acids. However, as the research targets become more and more complicated, the barriers of using NMR techniques become more and more severe. My dissertation focuses on two of those most difficult cases, one is about the backbone chemical shift assignment of intrinsically disordered proteins, and the other is about using four-dimensional diagonal suppressed methyl-methyl NOESY experiment to get methyl-methyl NOE information of large proteins.Intrinsically disordered proteins can perform their functions without forming a stable three-dimensional structure. Due to their large structural flexibilities, intrinsically disordered proteins are hard to crystallize, which makes NMR spectroscopy become the major technology used to study intrinsically disordered proteins. However, the peak overlap in NMR spectrums greatly hinders the process of chemical shift assignment of intrinsically disordered proteins. In the second chapter of this dissertation, we described the construction of two pairs of four-dimensional experiments-HNCACB/HN(CO)CACB&HA(CA)CO(CA)NH/HA(CA)CONH, and use them to get the backbone chemical shift assignment of the N-terminal sequence of a human intrinsically disordered protein-SKIP. By using non-uniform sampling, these two pairs of experiments canbe finished within11hours, which makes the assignment procedure quite efficient.Recently, new isotope labeling strategies have made it possible to use NMR spectroscopy to study proteins with high molecular weight. Methyl selective protonation is one of such newly developed labeling strategy. It brings in protonation in the methyl group of special amino acid residues, while leaves the background highly deuterated. This procedure can greatly improve the protein relaxation property, which at the same time restore the methyl-methyl NOE information. The methyl groups are often localized in hydrophobic cores of well folded proteins, which makes the methyl-methyl NOE information quite useful for the calculation of protein structures. In the third chapter of this dissertation, we provided a way to apply non-uniform sampling to NOESY experiment. NOESY-type experiments always contain strong diagonal peaks, which would generate strong artifacts when non-uniform sampling were applied. These artifacts maybe even stronger than the weak cross peaks, which would cause the loss of important NOE information. So, we developed a diagonal signal suppressed four-dimensional methyl-methyl NOESY experiment to apply ultrafast sampling. We tested this experiment on a42kDa E.coli protein-MBP, and achieve desired results.