title: Reconstitution and engineering of apoptotic protein interactions on the bacterial cell surface.
abstract: The interactions between pro- and anti-apoptotic members of the Bcl-2 class of proteins control whether a cell lives or dies, and the study of these protein-protein interactions has been an area of intense research. In this report, we describe a new tool for the study and engineering of apoptotic protein interactions that is based on the flow cytometric detection of these interactions on the surface of Escherichia coli. After validation of the assay with the well-studied interaction between the Bak(72-87) peptide and the anti-apoptotic protein Bcl-x(L), the effect of both increasing and decreasing Bak peptide length on Bcl-x(L) binding was investigated. Previous work demonstrated that the Bak(72-87) peptide also binds to the anti-apoptotic protein Bcl-2, albeit with lower binding affinity compared to Bcl-x(L). Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-x(L) and Bcl-2 equally well. Approximate binding affinity calculations on these peptide-protein complexes confirm the experimental observations. The flow cytometric assay was also used to screen a saturation mutagenesis library of Bak(72-87) variants for improved affinity to Bcl-x(L). The best variants obtained from this library exhibit an apparent K(d) to Bcl-x(L) 4-fold lower than that of wild-type Bak(72-87).
authors.affiliation: Department of Chemical Engineering, Princeton University, Princeton, NJ 08544, USA.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Sun
authors.forename: Diya M
authors.initials: DM
authors.lastname: Abdeljabbar
authors.forename: Nicole
authors.initials: N
authors.lastname: Clarke
authors.forename: Meghan L
authors.initials: ML
authors.lastname: Bellows
authors.forename: Christodoulos A
authors.initials: CA
authors.lastname: Floudas
authors.forename: A James
authors.initials: AJ
authors.lastname: Link
mesh_terms: D000818:Animals; D017209:Apoptosis; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D016296:Mutagenesis; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D010455:Peptides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; D014443:Tyrosine; D001120:Arginine