Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap

Scope and mechanism

Thomas J. Magliery, Christopher G M Wilson, Weilan Pan, Dennis Mishler, Indraneel Ghosh, Andrew D. Hamilton, Lynne Regan

Research output: Contribution to journalArticle

298 Citations (Scopus)

Abstract

Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (KD ≈ 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.

Original languageEnglish (US)
Pages (from-to)146-157
Number of pages12
JournalJournal of the American Chemical Society
Volume127
Issue number1
DOIs
StatePublished - Jan 12 2005

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Green Fluorescent Proteins
Proteins
Proteomics
Leucine Zippers
Protein Binding
Escherichia coli
Plasmids
Fusion reactions
Fasteners
Ligands
Peptides
Kinetics
Experiments

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap : Scope and mechanism. / Magliery, Thomas J.; Wilson, Christopher G M; Pan, Weilan; Mishler, Dennis; Ghosh, Indraneel; Hamilton, Andrew D.; Regan, Lynne.

In: Journal of the American Chemical Society, Vol. 127, No. 1, 12.01.2005, p. 146-157.

Research output: Contribution to journalArticle

Magliery, Thomas J. ; Wilson, Christopher G M ; Pan, Weilan ; Mishler, Dennis ; Ghosh, Indraneel ; Hamilton, Andrew D. ; Regan, Lynne. / Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap : Scope and mechanism. In: Journal of the American Chemical Society. 2005 ; Vol. 127, No. 1. pp. 146-157.
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