Re-engineering a split-GFP reassembly screen to examine RING-domain interactions between BARD1 and BRCA1 mutants observed in cancer patients.
Identification of protein-protein interactions is critical for understanding protein function and regulation. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2-hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly, also called Bimolecular Fluorescence Complementation (BiFC), is especially attractive because the GFP chromophore forms spontaneously on protein folding in virtually every cell type tested. However, cellular fluorescence evolves slowly in bacteria and fails to evolve at all for some interactions. We aimed to use split-GFP reassembly to examine the determinants of association for a heterodimeric four-helix bundle, and we chose the N-terminal RING domains of BARD1 and the tumor suppressor BRCA1 as our test system. The wild-type interaction failed to give fluorescence with the split sg100 GFP variant. We found that split folding-reporter GFP (a hybrid of EGFP and GFPuv) evolves fluorescence much faster (overnight) with associating peptides and also evolves fluorescence for the BRCA1/BARD1 wild-type pair. Six cancer-associated BRCA1 interface mutants were examined with the system, and only two resulted in a significant reduction in complex reassembly. These results are generally in accord with Y2H studies, but the differences highlight the utility of complementary approaches. The split frGFP system may also be generally useful for other proteins and cell types, as the split-Venus system has proven to be in mammalian cells.
Sarkar
Mohosin
M
Department of Chemistry, The Ohio State University, 100 W. 18th Ave., Columbus, OH 43210-1185, USA.
Magliery
Thomas J
TJ
D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D019313:BRCA1 Protein; D050505:Mutant Proteins; D025521:Tumor Suppressor Proteins; D049452:Green Fluorescent Proteins; C102449:BARD1 protein, human; D044767:Ubiquitin-Protein Ligases