A high efficiency strategy for binding property characterization of peptide-binding domains.
A large proportion of protein-protein interactions is mediated by families of peptide-binding domains. Comprehensive characterization of each of these domains is critical for understanding the mechanisms and networks of protein interaction at the domain level. However, existing methods are all based on large scale screenings for each domain that are inefficient to deal with hundreds of members in major domain families. We developed a systematic strategy for efficient binding property characterization of peptide-binding domains based on high throughput validation screening of a specialized candidate ligand library using yeast two-hybrid mating array. Its outstanding feature is that the overall efficiency is dramatically improved compared with that of traditional screening, and it will be higher as the system cycles. PDZ domain family was first used to test the strategy. Five PDZ domains were rapidly characterized. Broader binding properties were identified compared with other methods, including novel recognition specificities that provided the basis for major revision of conventional PDZ classification. Several novel interactions were discovered, serving as significant clues for further functional investigation. This strategy can be easily extended to a variety of peptide-binding domains as a powerful tool for comprehensive analysis of domain binding property in proteomic scale.
Song
Eli
E
Proteomics Research Center, National Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences/Peking Union Medical College, 100005 Beijing, China.
Gao
Shijuan
S
Tian
Rui
R
Ma
Sucan
S
Huang
Haiming
H
Guo
Jiayan
J
Li
Yingna
Y
Zhang
Ling
L
Gao
Youhe
Y
D000818:Animals; D006801:Humans; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary / Q000502:physiology; D020798:Two-Hybrid System Techniques
D019151:Peptide Library; D010455:Peptides