Open Access Dissertation
Date of Award
Christine S. Chow
In the development of finding a peptide targeting H69 of 23S rRNA in bacterial ribosomes, phage display was employed at pH 5.5, a buffer condition previously reported of H69 preferring a closed conformation. After sequencing, several peptides were chosen through sequence alignment, followed by preparation using solid-phase peptide synthesis. The peptides were characterized using MALDI-TOF and purified with HPLC. A truncated peptide TARHIY was selected from FID assay. Through binding studies using ESI-MS, SPR, BLItz, and NMR, the binding properties of the peptide to H69 were determined, such as binding affinity, stoichiometry, and interaction site. The peptide exhibited moderate binding affinity towards H69 (apparent Kd~10 µM) using ESI-MS, SPR, and BLItz, and the methods correspond to each other, however, no selectivity towards a buffer condition or RNA type was detected. Data obtained from ESI-MS suggested dimeric binding at higher concentrations, which was explored in the later part of the research. The interaction site of the peptide towards H69 was explored using NMR, which was in the loop region of H69.
Multimeric binding of peptides to H69 was explored by using dimeric peptides. Dimeric peptides with the same or different sequences on amino groups of lysine were prepared using solid-phase peptide synthesis. Improved binding affinity (apparent Kd~1 µM) of the dimer towards H69 compared to the monomer peptide were obtained using ESI-MS and BLItz. The binding affinity of the dimer TT was comparable to neomycin, a known aminoglycoside, while reverse dimer TY, exhibited decrease in binding affinity, suggesting the N-terminus plays an important role in binding, and that the 1:2 RNA:peptide complexes observed in ESI-MS spectra were not solely due to aggregation. An overall conformational change was observed with dimer TT using NMR, which was also comparable to neomycin. Further studies will help elucidate the actual binding mode of peptide TARHIY towards H69. These results suggest the possibility of multimeric binding should be taken into consideration with peptides selected from phage display.
Seo, Hyosuk, "Ligand Binding Studies Of A Peptide Targeting Helix 69 Of 23s Rrna In Bacterial Ribosomes" (2017). Wayne State University Dissertations. 1874.