Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface

Jingjing Sun, Diya M. Abdeljabbar, Nicole Clarke, Meghan L. Bellows, Christodoulos A. Floudas, A. James Link

Research output: Contribution to journalArticle

10 Scopus citations

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-xL, the effect of both increasing and decreasing Bak peptide length on Bcl-xL 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-xL. Here, we demonstrate that a slightly longer Bak peptide corresponding to amino acids 72-89 of Bak binds Bcl-xL 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-xL. The best variants obtained from this library exhibit an apparent Kd to Bcl-xL 4-fold lower than that of wild-type Bak(72-87).

Original languageEnglish (US)
Pages (from-to)297-305
Number of pages9
JournalJournal of Molecular Biology
Volume394
Issue number2
DOIs
StatePublished - Nov 27 2009

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

Keywords

  • Bcl-2 proteins
  • apoptosis
  • cell surface display
  • protein-protein interactions

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