Bcl-2 protein family members play crucial roles in programmed cell death. The fate of the cell relies on interactions between anti- and pro-apoptotic members of the Bcl-2 family. We have developed a new tool to study these interactions by displaying a 16-mer peptide containing the Bcl-2 homology 3 (BH3) region of the pro-apoptotic members on the E. coli cell surface, and examining their binding affinity toward anti-apoptotic proteins in solution through fluorescent labeling and flow cytometry. We present quantitative data for a near-complete set of 17 BH3 domains from the human genome binding to each of the 5 anti-apoptotic Bcl-2 family members. This dataset provides insights into quantitative structure-activity relationship analysis of the BH3 domains. A thorough truncation study of the most potent peptide, Bim, was carried out in order to see its shortest functional length. Via genetic engineering, libraries of BH3 peptides mutated at 4 key hydrophobic positions were created and screened for improved affinity. A Bak BH3 variant was found that bound the anti-apoptotic protein Bcl-xL with an affinity 13-fold high than the wild-type peptide. Variants with improved affinity toward one protein were also tested with the other 4 anti-apoptotic proteins to see if the affinity improvement could be transferred onto another target. Highly avid BH3 peptide variants that bind to multiple anti-apoptotic proteins have strong potential to serve as anti-cancer therapeutics.
This preliminary program is subject to change.
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Measuring and Engineering the Bcl-2 Family Interactome Using Bacterial Surface Display
Bcl-2 protein family members play crucial roles in programmed cell death. The fate of the cell relies on interactions between anti- and pro-apoptotic members of the Bcl-2 family. We have developed a new tool to study these interactions by displaying a 16-mer peptide containing the Bcl-2 homology 3 (BH3) region of the pro-apoptotic members on the E. coli cell surface, and examining their binding affinity toward anti-apoptotic proteins in solution through fluorescent labeling and flow cytometry. We present quantitative data for a near-complete set of 17 BH3 domains from the human genome binding to each of the 5 anti-apoptotic Bcl-2 family members. This dataset provides insights into quantitative structure-activity relationship analysis of the BH3 domains. A thorough truncation study of the most potent peptide, Bim, was carried out in order to see its shortest functional length. Via genetic engineering, libraries of BH3 peptides mutated at 4 key hydrophobic positions were created and screened for improved affinity. A Bak BH3 variant was found that bound the anti-apoptotic protein Bcl-xL with an affinity 13-fold high than the wild-type peptide. Variants with improved affinity toward one protein were also tested with the other 4 anti-apoptotic proteins to see if the affinity improvement could be transferred onto another target. Highly avid BH3 peptide variants that bind to multiple anti-apoptotic proteins have strong potential to serve as anti-cancer therapeutics.