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Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.

ABSTRACT: Split reporter-based bioluminescence imaging is a useful strategy for studying protein-protein as well as other intracellular interactions. We have used a combinatorial strategy to identify a novel split site for firefly luciferase with improved characteristics over previously published split sites. A combination of fragments with greater absolute signal with near-zero background signals was achieved by screening 115 different combinations. The identified fragments were further characterized by using five different interacting protein partners and an intramolecular folding strategy. Cell culture studies and imaging in living mice was performed to validate the new split sites. In addition, the signal generated by the newly identified combination of fragments (Nfluc 398/ Cfluc 394) was compared with different split luciferase fragments currently in use for studying protein-protein interactions and was shown to be markedly superior with a lower self-complementation signal and equal or higher postinteraction absolute signal. This study also identified many different combinations of nonoverlapping and overlapping firefly luciferase fragments that can be used for studying different cellular events such as subcellular localization of proteins, cell-cell fusion, and evaluating cell delivery vehicles, in addition to protein-protein interactions, both in cells and small living animals.

SUBMITTER: Paulmurugan R

PROVIDER: S-EPMC3198827 | biostudies-literature | 2007 Mar

REPOSITORIES: biostudies-literature

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Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.

Paulmurugan Ramasamy R Gambhir Sanjiv S SS

Analytical chemistry 20070213 6

Split reporter-based bioluminescence imaging is a useful strategy for studying protein-protein as well as other intracellular interactions. We have used a combinatorial strategy to identify a novel split site for firefly luciferase with improved characteristics over previously published split sites. A combination of fragments with greater absolute signal with near-zero background signals was achieved by screening 115 different combinations. The identified fragments were further characterized by ...[more]

PMID: 17295448

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Project description:Hsp90 requires cochaperone Cdc37 to load its clients to the Hsp90 superchaperone complex. The purpose of this study was to utilize split Renilla luciferase protein fragment-assisted complementation (SRL-PFAC) bioluminescence to study the full-length human Hsp90-Cdc37 complex and to identity critical residues and their contributions for Hsp90/Cdc37 interaction in living cells. SRL-PFAC showed that full-length human Hsp90/Cdc37 interaction restored dramatically high luciferase activity through Hsp90-Cdc37-assisted complementation of the N and C termini of luciferase (compared with the set of controls). Immunoprecipitation confirmed that the expressed fusion proteins (NRL-Hsp90 and Cdc37-CRL) preserved their ability to interact with each other and also with native Hsp90 or Cdc37. Molecular dynamic simulation revealed several critical residues in the two interaction patches (hydrophobic and polar) at the interface of Hsp90/Cdc37. Mutagenesis confirmed the critical residues for Hsp90-Cdc37 complex formation. SRL-PFAC bioluminescence evaluated the contributions of these critical residues in Hsp90/Cdc37 interaction. The results showed that mutations in Hsp90 (Q133A, F134A, and A121N) and mutations in Cdc37 (M164A, R167A, L205A, and Q208A) reduced the Hsp90/Cdc37 interaction by 70-95% as measured by the resorted luciferase activity through Hsp90-Cdc37-assisted complementation. In comparison, mutations in Hsp90 (E47A and S113A) and a mutation in Cdc37 (A204E) decreased the Hsp90/Cdc37 interaction by 50%. In contrast, mutations of Hsp90 (R46A, S50A, C481A, and C598A) and mutations in Cdc37 (C54S, C57S, and C64S) did not change Hsp90/Cdc37 interactions. The data suggest that single amino acid mutation in the interface of Hsp90/Cdc37 is sufficient to disrupt its interaction, although Hsp90/Cdc37 interactions are through large regions of hydrophobic and polar interactions. These findings provides a rationale to develop inhibitors for disruption of the Hsp90/Cdc37 interaction.

Project description:A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Dataset Information

Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.

Publications

Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions.

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