Development and application of methodology for rapid NMR data collection and protein structure determination
Parish, David Michael
MetadataShow full item record
This thesis focuses on the development and application of methodology for rapid NMR data collection and protein structure determination. In chapter 1, simultaneously cycled (SC) NMR is introduced and exemplified by implementing without readout gradients a set of 2D [ 1 H, 1 H] SC Exclusive COSY (E.COSY) NMR experiments and with readout gradients a 2D [ 1 H, 1 H] double quantum filtered (DQF) COSY experiment. Spatially selective 1 H r.f. pulses are applied as composite pulses such that n steps of the respective cycles are effected simultaneously in n slices of the sample, thus reducing total acquisition time by a factor of n. In chapter 2, the structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium (NESG target StR70) was determined by NMR, refined using residual dipolar coupling constraints and compared to the X-ray structure of Q8ZP25_SALTY and the NMR structure of homologous protein HYAE_ECOLI. Protein Q8ZP25_SALTY belongs to Pfam PF07449, which itself belongs to the 'thioredoxin-like clan'. However, protein Q8ZP25_SALTY and the other proteins of Pfam PF07449, do not contain the Cys-X-X-Cys active site sequence motif of thioredoxin. The structures presented here exhibit the expected thioredoxin-like fold and support biochemical data suggesting that members of Pfam family PF07449 specifically interact with Tat signal peptides involved in hydrogenase assembly. In chapter 3, the development of a hardware and software infrastructure designed specifically to support high throughput NMR protein structure determination for structural genomics is described. In addition, a "consensus run" protocol is detailed which uses common results from two disparate programs for automated NMR structure determination, namely, CYANA and AUTOSTRUCTURE, to minimize errors in initial NOESY peak assignments. The high throughput infrastructure and consensus run have supported the determination of 47 protein structures to date. Finally, in chapter 4, the relaxation agent and gadolinium chelate gadoversetamide is used to affect the T 1 and T 2 relaxation times of amide and methyl protons in proteins in aqueous solution so as to map protein surfaces. The protocol is explored to distinguish surface and buried residues in order to identify homo-dimer interfaces.