TY - JOUR
T1 - Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell Surface
AU - Sun, Jingjing
AU - Abdeljabbar, Diya M.
AU - Clarke, Nicole
AU - Bellows, Meghan L.
AU - Floudas, Christodoulos A.
AU - Link, A. James
N1 - Funding Information:
We are grateful to Patrick Daugherty (University of California, Santa Barbara) for the gift of pB33eCPX-SApep and to Michael Diehl (Rice University) for pMON-BirA. We also thank Christina DeCoste for assistance with cell sorting. This work was supported by Princeton University startup funds to A.J.L. C.A.F. acknowledges support from the National Science Foundation ( CTS-0426691 ), National Institutes of Health ( R01 GM52032 ), and U.S. Environmental Protection Agency ( GAD R832721-010 ).
PY - 2009/11/27
Y1 - 2009/11/27
N2 - 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).
AB - 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).
KW - Bcl-2 proteins
KW - apoptosis
KW - cell surface display
KW - protein-protein interactions
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U2 - 10.1016/j.jmb.2009.09.023
DO - 10.1016/j.jmb.2009.09.023
M3 - Article
C2 - 19766123
AN - SCOPUS:70350536769
SN - 0022-2836
VL - 394
SP - 297
EP - 305
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 2
ER -