Methodology And Applications Of High Resolution NMR In Protein Studies

This thesis focuses on the methodology and applications of high resolution NMR spectroscopy in protein study. The scope of the thesis includes some development in protein NMR techniques, protein dynamics study and protein-ligand interaction study.1. The recent development in this field especially in the study of protein-ligand intraction was reviewed. And the basic theory on which the thesis stands was summed up.2. Dynamic studies of Trichosanthin (TCS): to alleviate the problem of serious resonance overlap in the solution structure determination of TCS using 15N/13C labeled sample, a Clean SEA-HSQC experiment has been developed based on the SEA-TROSY experiment. This method selectively observes solvent exposed amide protons without contamination with exchange-relayed NOE contributions from fast exchanging hydroxyl or amine protons and longitudinal relaxation contributions in non-deuterated proteins or protein-ligand complexes. Compared with the water accessibility calculated from the crystal structure of TCS, the result of Clean SEA-HSQC study of TCS revealed that the solution structure of TCS is quite similar to its crystal structure. In addition, from the studies of the 15N relaxation data, the backbone dynamics information on residue basis was obtained.3. The competitive binding of two ligands, ibuprofen and salicylic acid, to human serum albumin (HSA) was studied using NMR chemical shift and relaxation measurments. It is demonstrated that the two ligands shared certain low-affinity binding sites on the HSA molecule, in addition to the same high-affinity binding site of AIII. The competitive binding causes decrement of the 1H relaxation rates (R1) and down-field drift of chemical shift of one ligand when the concentration of the other one was increased in the solution containing HSA. For a fast exchange system, value of the observed 1H relaxation rate is assumed to be the weighted average of the ligand at free and bound states. This leads to quantitative derivation of the concentrations of the free and bound ligands. The number of...