Detecting Protein-Protein Interactions with a Green Fluorescent Protein Fragment Reassembly Trap: Scope and Mechanism
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- 作者：Thomas J. Magliery ; Christopher G. M. Wilson ; Weilan Pan ; Dennis Mishler ; Indraneel Ghosh ; Andrew D. Hamilton ; and Lynne Regan
- 刊名：Journal of the American Chemical Society
- 出版年：2005
- 出版时间：January 12, 2005
- 年：2005
- 卷：127
- 期：1
- 页码：146 - 157
- 全文大小：603K
- 年卷期：v.127,no.1(January 12, 2005)
- ISSN：1520-5126
文摘
Identification of protein binding partners is one of the key challenges of proteomics. We recentlyintroduced a screen for detecting protein-protein interactions based on reassembly of dissected fragmentsof green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichiacoli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a libraryof antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (KD 
1mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that thescreen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognatefrom noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experimentsdemonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guidethe further use of this screen in proteomic analysis. Additionally, this work gives insight into the positionalinequivalence of stabilizing interactions in antiparallel coiled coils.
1mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that thescreen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognatefrom noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experimentsdemonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guidethe further use of this screen in proteomic analysis. Additionally, this work gives insight into the positionalinequivalence of stabilizing interactions in antiparallel coiled coils.