The Bcl-2 family of proteins regulates apoptosis at the level of mitochondrial permeabilization. Pro-death members of the family, including Bak and Bax, initiate apoptosis, whereas pro-survival members such as Bcl-xL and Mcl-1 antagonize the function of Bak and Bax via heterodimeric interactions. These heterodimeric interactions are primarily mediated by the binding of the helical amphipathic BH3 domain from a prodeath protein to a hydrophobic cleft on the surface of the prosurvival protein. Since high levels of pro-survival Bcl-2 proteins are present in many cancers, peptides corresponding to pro-death BH3 domains hold promise as therapeutics.Here we apply a highthroughput flow cytometry assay to engineer the Bak BH3 domain for improved affinity toward the pro-survival proteins Bcl-xL and Mcl-1. Two strategies, engineering the hydrophobic face of the Bak BH3 peptide and increasing its overall helicity, are successful in identifying Bak BH3 variants with improved affinity to Bcl-xL and Mcl-1. Hydrophobic face engineering of the Bak BH3 peptide led to variants with up to a 15-fold increase in affinity for Bcl-xL and increased specificity toward Bcl-xL. Engineering of the helicity of Bak BH3 led to modest (3- to 4-fold) improvements in affinity with retention of promiscuous binding to all pro-survival proteins. HeLa cell killing studies demonstrate that the affinity matured Bak BH3 variants retain their expected biological function.
|Original language||English (US)|
|Number of pages||10|
|Journal||ACS Synthetic Biology|
|State||Published - Mar 16 2012|
All Science Journal Classification (ASJC) codes
- Biomedical Engineering
- Biochemistry, Genetics and Molecular Biology (miscellaneous)