Application and development of multi-dimensional NMR spectroscopic techniques to study protein structure in solution.

摘要

With the advent of multidimensional heteronuclear-edited and filtered NMR experiments, the field of three-dimensional structure determination by NMR has again and again increased in scope, making it possible to move the technology beyond the approximately 10kDa limit inherent to conventional two-dimensional NMR to system up to potentially 35 to 40 kDa. This thesis reported innovative pulse sequences to improve the sensitivity and resolution of NMR spectroscope for protein structure determination, application of new pulse sequences to solving structure of eIF-4e, a 30 kDa protein playing key role in translation initiation, and solution structure, plus dynamics study, of N-terminal region of SV40 large T Antigen.; A sensitive triple resonance experiment HN(CA)CO was designed to take advantage of the decreased decay rate of α-carbon and selective carbon pulses which can be specifically applied to either α-carbon or β-carbon instead of the entire carbon channel. In addition, an innovative protein preparation procedure was developed to replace protons in a protein with deuterium. More room for sensitivity improvement was identified as it is possible to use selective Cα pulse to minimize the carbon-carbon coupling in 13C-enriched protein.; EIF-4e is a master switch that controls eukaryotic translation as it binds mRNA m7G_pppN cap to initiate the cap dependent translation. We have solved the structure of the yeast eIF4e/m7G _pp complex, determined the position of the second nucleotide in a complex with m7G_pppA, and identified the 4E-BP binding site. EIF-4e has a curved eight-stranded anti-parallel β sheet, decorated with three helices on the convex face and three smaller helices inserted in connecting loops. The m7G of the cap is intercalated into a stack of tryptophans in the concave face. The 4E-BP binding site is located in a region encompassing one edge of the β-sheet, the adjacent helix and several regions of the non-regular secondary structure. It is adjacent to, but does not overlap the cap-binding site.; Complete backbone assignment has been obtained for the N-terminal (1–135) of SV40 Large T Antigen. The secondary structure has been characterized using a Carbon shifts and medium range NOEs. The protein consists of four helices connected by three loop regions. The pRb family proteins association motif LxCxE and nuclear localization sequence KKKRK are located in the unstructured region. The high-resolution structure of N-terminal 1–82 fragment was determined on the basis of 1,074 NOE upper distance limits and 61 dihedral angle constraints. It contains three well-defined helices comprising residue 9–18, 28–41, and 51–69, as well as a fourth helix, consisting of residues 2–5, which has well defined secondary structure but relatively loosely defined side chain conformation. The conformation of tripeptide HPD, a conserved motif among DnaJ domain proteins, is stabilized by the hydrophobic contact among Pro43, Phe41, and Met55 side-chains with the hydrophilic side-chains of His42 and Asp44 extending out toward solvent. The structure supports the hypothesis that this highly conserved tripeptide could play a key role in the interaction of T Antigen with Hsp70 family proteins.