Methods to identify novel α-helical peptides that bind to LMO4

Abstract

Protein-protein interactions (PPIs) play an essential role in regulating cells. LIM Domain-Only protein 4 (LMO4) is a transcriptional co-regulatory protein that functions entirely through PPIs. It contributes to developmental processes and the pathogenesis of breast cancer, although the mechanisms are only partially known. The development of inhibitors of LMO4 could form useful research tools for understanding these mechanisms and may form the basis of new breast cancer therapies. Previously, the Matthews Laboratory has developed peptide inhibitors of LMO4 based on natural binding partners (β-strand peptides). The hypotheses is that α-helical peptides can be more specific than β-strand peptides, it should be possible to generate α-helical peptide binders of LMO4 using natural α-helices as templates. The aims were to develop two methods to identify novel α-helical peptides that bind to LMO4: a split EGFP complementation (spEGFP) system providing a high through-put method for the initial screening of a library of α-helical peptides; and, a Yeast Two Hybrid Competition Assay (Y2HCA) as an orthogonal method to validate hits and provide an assessment of their relative binding affinities. For the Y2HCA, a new construct was added to create a series of constructs that relatively assess affinities of weakly binding peptides. The spEGFP system was expanded, introducing controls and a construct to screen a library of α-helical peptides for affinity to LMO4LIM1. Naturally occurring α-helices were considered and tested as potential templates. Chemical transformation and DNA extraction protocols for BL-21(DE3) cells were developed to enable efficient library expression and screening. This thesis provides mechanisms to identify α-helical peptides that bind to LMO4 and the ground work for a high-throughput library screen.