Open Access Dissertation
Date of Award
Christine S. Chow
Peptides binding to helix 69 of domain IV or residues 1906-1924, of 23 S rRNA of E.coli were being selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through four rounds was being followed. After sequencing phage clones of the fourth round, two peptide sequences dominated the phage pool, STYTSVS and NQVANHQ. The later sequence was a unique sequence since this sequence contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. Phage-display methodology demonstrated the rapid feasibility of identification, and isolation of small peptides that bind to 23 S rRNA in an effort to discover new RNA-binding motifs that have potential therapeutic applications.
For evaluating the preliminary binding affinity of these peptides with H69, fluorescence assays were applied. For this assay, the fluorescence intensity of the NQVANHQ Tentagel beads was observed to be higher than STYTSVS Tentagel beads indicating that peptide NQVANHQ is having higher affinity for H69 as compared to STYTSVS peptide. But the higher binding affinity of the NQVANHQ peptide was further validated with more sensitive method of electrospray ionization (ESI) mass spectroscopy. The apparent dissociation constant (Kd) obtained for H69 and NQVANHQ-NH2 peptide was in the low micromolar range (11 µM). This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA (1 to 10 µM).
The ESI-MS experiments with H69 variant UUU RNA and peptide NQVANHQ-NH2 gave the relative dissociation constant (Kd) at 1:1 stoichiometry as 19 µM. The higher value of Kd for this complex revealed that the presence of all three pseudouridine residues positively contributed towards binding of this peptide to H69. Consecutively, to learn about the role of individual pseudouridines at position 1911 and 1915 towards binding of the peptide, the ESI-MS experiments were performed with two H69 variants,UYY and YUY. The apparent dissociation constants (Kd) for the 1:1 complex for these two RNAs decreased by 2.5-fold showing that peptide binding site is located at or near the loop region containing the pseudouridines at positions 1911 and 1915. In addition, the effect of pH on the complex formation of H69 and UUU RNA with NQVANHQ-NH2 peptide was studied at two different pH values of 7.0 and 5.2. There was three-fold decrease of the apparent dissociation constant for the 1:1 complex of RNA and the peptide indicating that either protonation of the RNA or the peptide structure influenced this change in binding of the two species.
The specificity of the peptide for H69 was tested with related RNA such as human H69 and unrelated RNAs such as helix 31 and A-site rRNA. The peptide showed three-fold lower affinity than the target H69 RNA for all these RNAs suggesting that the peptide has features for developing it as a lead compound for novel antimicrobial.
Kaur, Moninderpal, "Isolation And Analysis Of Peptides Binding To Helix 69 Of E. Coli 23 S Rrna From M13 Phage Display" (2011). Wayne State University Dissertations. 196.