Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism.
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.
Magliery
Thomas J
TJ
Department of Molecular Biophysics & Biochemistry, Yale University, P.O. Box 208114, New Haven, Connecticut 06520-8114, USA.
Wilson
Christopher G M
CG
Pan
Weilan
W
Mishler
Dennis
D
Ghosh
Indraneel
I
Hamilton
Andrew D
AD
Regan
Lynne
L
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016350:Leucine Zippers; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins