|Journal of the American Chemical Society (2005) 127(1):146-57|
|Northeast Structural Genomics Consortium|
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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 (K(D) approximately 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.
|chemistry methods genetics |
|Green Fluorescent Proteins Models, Molecular Escherichia coli Molecular Sequence Data Recombinant Fusion Proteins Biophysical Phenomena Leucine Zippers Kinetics Proteomics Protein Folding Plasmids Amino Acid Sequence Biophysics Peptide Fragments |
|237 (Last update: 03/25/2017 12:07:22pm)|
|J Am Chem Soc. 2005 Jan 12;127(1):146-57.|