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| Status |
Public on Aug 17, 2020 |
| Title |
Uncovering the basis of protein-protein interaction specificity with a combinatorially complete library |
| Organism |
synthetic construct |
| Experiment type |
Other
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| Summary |
Protein-protein interaction specificity is often accomplished by molecular recognition and mediated by a small set of interfacial residues. However, the extent to which these residues serve positive specificity roles (encourage interaction with the cognate protein) or negative specificity roles (destabilize interaction with the non-cognate protein) is poorly understood. To systematically dissect these roles, we built a library of interface variants using a bacterial toxin-antitoxin system, Mesorhizobium opportunistum ParD3-ParE3, as a model. The ParD3 antitoxin is specific for its cognate toxin ParE3 and does not neutralize the non-cognate toxin ParE2. We built a saturation mutagenesis library at three key interface positions in ParD3 and measured the ability of each variant to neutralize ParE3 and ParE2 via a 10-hour bulk selection assay. A fitness score was assigned to each variant in the presence of each toxin based on the relative expansion of the variant during the experiment (between t = 0 and t = 600 min).
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| Overall design |
Antitoxin variant frequency was measured for two toxins (M. opportunistum ParE3 and ParE2) upon toxin induction (t = 0) and after 10 hours of toxin and antitoxin library co-expression (t = 600). Two replicates were studied for each toxin strain. Selection in the presence of toxin expression was performed independently for each replicate.
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| Contributor(s) |
Lite TV, Grant RA, Nocedal I, Guo MS, Laub MT |
| Citation(s) |
33107822 |
| |
| Submission date |
Jul 06, 2020 |
| Last update date |
Nov 24, 2020 |
| Contact name |
Thuy-Lan Lite |
| Organization name |
Massachusetts Institute of Technology
|
| Street address |
31 Ames Street
|
| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platforms (1) |
| GPL19424 |
Illumina NextSeq 500 (synthetic construct) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA644451 |
| SRA |
SRP270411 |
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