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Isolation and analysis of peptides b...

Kaur, Moninderpal.

Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display.
Author:	Kaur, Moninderpal.
Description:	256 p.
Notes:	Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: 2093.
Notes:	Adviser: Christine S. Chow.
Contained By:	Dissertation Abstracts International72-04B.
Subject:	Biology, Microbiology.
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3440300
ISBN:	9781124475004

Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display.
Kaur, Moninderpal.

Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display. - 256 p.

Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: 2093.

Thesis (Ph.D.)--Wayne State University, 2011.

Peptides binding to helix 69 (H69) of domain IV or residues 1906 to1924 of E. coli 23 S rRNA were selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through multiple rounds was 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 that contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. The phage-display methodology demonstrated the feasibility of rapid ligand 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.

ISBN: 9781124475004Subjects--Topical Terms:

226920
Biology, Microbiology.

Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display.
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100 1 $a Kaur, Moninderpal. $3 530971
245 1 0 $a Isolation and analysis of peptides binding to helix 69 of E. coli 23 S rRNA from M13 phage display.
300 $a 256 p.
500 $a Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: 2093.
500 $a Adviser: Christine S. Chow.
502 $a Thesis (Ph.D.)--Wayne State University, 2011.
520 $a Peptides binding to helix 69 (H69) of domain IV or residues 1906 to1924 of E. coli 23 S rRNA were selected from a heptapeptide phage library. An experimental system including biotin labeling of RNA and then affinity selection through multiple rounds was 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 that contained an abundance of amino acid residues that are also present in the ribosome recycling factor, RRF, and known to make contacts with H69. The phage-display methodology demonstrated the feasibility of rapid ligand 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.
520 $a For evaluating the preliminary binding affinity of these peptides with H69, fluorescence assays were applied. For this assay, the fluorescence intensity of NQVANHQ Tentagel beads was observed to be higher than STYTSVS Tentagel beads, indicating that peptide NQVANHQ has a higher affinity for H69 as compared to STYTSVS. The higher binding of the NQVANHQ peptide was further validated with a more sensitive method known as electrospray ionization (ESI) mass spectroscopy (MS). The apparent dissociation constant (Kd) obtained for H69 and NQVANHQ-NH2 was in the low micromolar range (11 +/- 1 microM) at pH 7.0 in 150 mM ammonium acetate buffer. This value is comparable to that of aminoglycoside antibiotics binding to the A-site RNA and H69 (1 to 10 microM).
520 $a The ESI-MS experiments with H69 variant UUU RNA and peptide NQVANHQ-NH 2 gave a relative dissociation constant (Kd) at 1:1 stoichiometry as 19 +/- 2 microM at pH 7.0. The higher value of Kd for this complex revealed that the presence of pseudouridine residues positively contributes towards binding of this peptide to H69. Consequently, to learn about the role of individual pseudouridines at positions 1911 and 1915 towards binding of the peptide, ESI-MS experiments were performed with two H69 variants, UPsiPsi and PsiUPsi. The apparent dissociation constants (Kds) for the 1:1 complex for these two RNAs decreased by 2.5-fold, suggesting that peptide binding site is located at or near the loop region of H69, which contains 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 was studied at two different values (7.0 and 5.2). There was a threefold 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.
520 $a 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 for all these RNAs compared to the target H69, suggesting that the peptide has features that are desirable for developing it as a lead compound for novel antimicrobials.
590 $a School code: 0254.
650 4 $a Biology, Microbiology. $3 226920
650 4 $a Chemistry, Analytical. $3 224793
650 4 $a Chemistry, Biochemistry. $3 226900
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690 $a 0486
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710 2 $a Wayne State University. $b Chemistry. $3 530972
773 0 $t Dissertation Abstracts International $g 72-04B.
790 1 0 $a Chow, Christine S., $e advisor
790 1 0 $a Feig, Andrew L. $e committee member
790 1 0 $a Verani, Claudio $e committee member
790 1 0 $a Dutta, Aloke $e committee member
790 $a 0254
791 $a Ph.D.
792 $a 2011
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3440300