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Simplified proteomics approach to discover protein-ligand interactions.

ABSTRACT: Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule. Here, we report a surprisingly simple version of energetics-based target identification, which only requires ion exchange chromatography, SDS PAGE, and minimal use of mass spectrometry. The complexity of a proteome is reduced through fractionation by ion exchange chromatography. Urea-induced unfolding of proteins in each fraction is then monitored by the significant increase in proteolytic susceptibility upon unfolding in the presence and the absence of a ligand. Proteins showing a different degree of unfolding with the ligand are identified by SDS PAGE followed by mass spectrometry. Using this approach, we identified ATP-binding proteins in the Escherichia coli proteome. In addition to known ATP-binding proteins, we also identified a number of proteins that were not previously known to interact with ATP. To validate one such finding, we cloned and purified phosphoglyceromutase, which was not previously known to bind ATP, and confirmed that ATP indeed stabilizes this protein. The combination of fractionation and pulse proteolysis offers an opportunity to investigate protein-drug or protein-metabolite interactions on a proteomic scale with minimal instrumentation and without modification of a molecule of interest.

SUBMITTER: Chang Y

PROVIDER: S-EPMC3631357 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Simplified proteomics approach to discover protein-ligand interactions.

Chang Youngil Y Schlebach Jonathan P JP VerHeul Ross A RA Park Chiwook C

Protein science : a publication of the Protein Society 20120723 9

Identifying targets of biologically active small molecules is an essential but still challenging task in drug research and chemical genetics. Energetics-based target identification is an approach that utilizes the change in the conformational stabilities of proteins upon ligand binding in order to identify target proteins. Different from traditional affinity-based capture approaches, energetics-based methods do not require any labeling or immobilization of the test molecule. Here, we report a su ...[more]

PMID: 22733688

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Project description:Bacterial surface ligands mediate interactions with the host cell during association that determines the specific outcome for the host-microbe association. The association begins with receptors on the host cell binding ligands on the microbial cell to form a partnership that initiates responses in both cells. Methods to determine the specific cognate partnerships are lacking. Determining these molecular interactions between the host and microbial surfaces are difficult, yet crucial in defining biologically important events that are triggered during association of the microbiome, and critical in defining the initiating signal from the host membrane that results in pathology or commensal association. In this study, we designed an approach to discover cognate host-microbe receptor/ligand pairs using a covalent cross-linking strategy with whole cells. Protein/protein cross-linking occurred when the interacting molecules were within 9-12 Å, allowing for identification of specific pairs of proteins from the host and microbe that define the molecular interaction during association. To validate the method three different bacteria with three previously known protein/protein partnerships were examined. The exact interactions were confirmed and led to discovery of additional partnerships that were not recognized as cognate partners, but were previously reported to be involved in bacterial interactions. Additionally, three unknown receptor/ligand partners were discovered and validated with in vitro infection assays by blocking the putative host receptor and deleting the bacterial ligand. Subsequently, Salmonella enterica sv. Typhimurium was cross-linked to differentiated colonic epithelial cells (caco-2) to discover four previously unknown host receptors bound to three previously undefined host ligands for Salmonella. This approach resulted in a priori discovery of previously unknown and biologically important molecules for host/microbe association that were casually reported to mediate bacterial invasion. The whole cell cross-linking approach promises to enable discovery of possible targets to modulate interaction of the microbiome with the host that are important in infection and commensalism, both of with initiate a host response.

Dataset Information

Simplified proteomics approach to discover protein-ligand interactions.

Publications

Simplified proteomics approach to discover protein-ligand interactions.

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