Abstract:
:Biochemical functions of proteins in cells frequently involve interactions with various ligands. Proteomic methods for the identification of proteins that interact with specific ligands such as metabolites, signaling molecules, and drugs are valuable in investigating the regulatory mechanisms of cellular metabolism, annotating proteins with unknown functions, and elucidating pharmacological mechanisms. Here we report an energetics-based target identification method in which target proteins in a cell lysate are identified by exploiting the effect of ligand binding on their stabilities. Urea-induced unfolding of proteins in cell lysates is probed by a short pulse of proteolysis, and the effect of a ligand on the amount of folded protein remaining is monitored on a proteomic scale. As proof of principle, we identified proteins that interact with ATP in the Escherichia coli proteome. Literature and database mining confirmed that a majority of the identified proteins are indeed ATP-binding proteins. Four identified proteins that were previously not known to interact with ATP were cloned and expressed to validate the result. Except for one protein, the effects of ATP on urea-induced unfolding were confirmed. Analyses of the protein sequences and structure models were also employed to predict potential ATP binding sites in the identified proteins. Our results demonstrate that this energetics-based target identification approach is a facile method to identify proteins that interact with specific ligands on a proteomic scale.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Liu PF,Kihara D,Park Cdoi
10.1016/j.jmb.2011.02.026subject
Has Abstractpub_date
2011-04-22 00:00:00pages
147-62issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(11)00171-9journal_volume
408pub_type
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