Covalent capture and identification of novel protein binding partners using an azide-tagged, photo-reactive stapled alpha helical p53 peptide

Thursday, November 07, 2013 — Poster Session II
12:00 p.m. – 2:00 p.m.	FAES Academic Center (Upper-Level Terrace)	NCI	CHEMCELL-25

Authors

A.L. Whiting
K.M. Headley
J.J. Mitala
Jr.
B.L. Morrison
K.A. Murray
F. Bernal

Abstract

Mutations in the DNA binding region of tumor suppressor p53 render it transcriptionally inactive (dominant negative) but can also manifest in transcriptionally-independent gain-of-function (GOF) effects, possibly due to mutp53 interacting with other, as yet, unknown targets. Identification of these targets would help to elucidate the pathways and vulnerabilities involved in mutp53 GOF. A hydrocarbon-stapled alpha helical peptide of the p53 transactivation domain (residues 14-29), SAH-p53-8, has been shown to strongly interact with known p53 targets that utilize this domain for binding (HDM2, Kd = 55 nM; HDMX, Kd = 2.3 nM ). Substitution of Leu26 for the unnatural amino acid benzoylphenylalanine (Bpa) results in a peptide that retains the biochemical properties of SAH-p53-8 (affinities of 38.1 nM and 58.3 nM, respectively) and can covalently capture known target proteins via photochemical reaction. The introduction of a versatile, biochemically-inert capping group to the N-terminus of this peptide opens up the ability to both visualize (via reaction with a fluorophore) and/or isolate (via reaction with an affinity tag) covalently bound unknown protein(s). This poster will detail both the chemical aspects of the tagged stapled peptide, as well as its use in protein capture in both recombinant systems and whole cells expressing p53 targets.