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1.  Using Unnatural Amino Acid Mutagenesis to Probe the Regulation of PRMT1† 
ACS chemical biology  2014;9(3):649-655.
Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS), however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-L-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-L-phenylalanine (pBpF) was incorporated at the Tyr291 position and crosslinking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.
PMCID: PMC4505744  PMID: 24358983
2.  Glucagon-Like Peptide-1 Receptor Ligand Interactions: Structural Cross Talk between Ligands and the Extracellular Domain 
PLoS ONE  2014;9(9):e105683.
Activation of the glucagon-like peptide-1 receptor (GLP-1R) in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM). Like other class B G protein-coupled receptors (GPCRs), the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX) to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R) were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R) peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R). In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.
PMCID: PMC4152014  PMID: 25180755
3.  Protein Conformation Ensembles Monitored by HDX Reveal a Structural Rationale for Abscisic Acid Signaling Protein Affinities and Activities 
Plants regulate growth and respond to environmental stress through abscisic acid (ABA) regulated pathways, and as such these pathways are of primary interest for biological and agricultural research. The ABA response is first perceived by the PYR/PYL/RCAR class of START protein receptors. These ABA activated receptors disrupt phosphatase inhibition of Snf1-related kinases (SnRKs) enabling kinase signaling. Here, insights into the structural mechanism of proteins in the ABA signaling pathway (the ABA receptor PYL2, HAB1 phosphatase, and two kinases, SnRK2.3 and 2.6) are discerned through hydrogen/deuterium exchange (HDX) mass spectrometry. HDX on the phosphatase in the presence of binding partners provides evidence for receptor-specific conformations involving the Trp385 ‘lock’ that is necessary for signaling. Furthermore, kinase activity is linked to a more stable closed conformation. These solution-based studies complement the static crystal structures and provide a more detailed understanding of the ABA signaling pathway.
PMCID: PMC3570687  PMID: 23290725
hydrogen deuterium exchange; mass spectrometry; protein conformation; abscisic acid; signaling pathways; kinase; phosphatase; phosphorylation; receptor; plant protein
4.  Thermodynamic Analysis of Protein-Ligand Binding Interactions in Complex Biological Mixtures using the Stability of Proteins from Rates of Oxidation (SPROX) Method 
Nature protocols  2012;8(1):148-161.
The detection and quantitation of protein-ligand binding interactions is critical in a number of different areas of biochemical research from fundamental studies of biological processes to drug discovery efforts. Described here is a protocol that can be used to identify the protein targets of biologically relevant ligands (e.g. drugs like tamoxifen or cyclosporin A) in complex protein mixtures such as cell lysates. The protocol utilizes quantitative, bottom-up, shotgun proteomics technologies (iTRAQ) with a covalent labeling technique, termed Stability of Proteins from Rates of Oxidation (SPROX). In SPROX, the thermodynamic properties of proteins and protein-ligand complexes are assessed using the hydrogen peroxide-mediated oxidation of methionine residues as a function of the chemical denaturant (e.g. guanidine Hydrochloride or urea) concentration. The proteome-wide SPROX experiments described here enable the ligand binding properties of hundreds of proteins to be simultaneously assayed in the context of complex biological samples. The proteomic capabilities of the protocol render it amenable to detection of both the on- and off-target effects of ligand binding.
PMCID: PMC3717606  PMID: 23257983
Protein-ligand; protein-drug; interaction; mass spectrometry; iTRAQ; shotgun proteomics; hydrogen peroxide; H2O2; methionine oxidation; denaturant; guanidine; urea; protein folding/unfolding
5.  HDX Workbench: Software for the Analysis of H/D Exchange MS Data 
Journal of the American Society for Mass Spectrometry  2012;23(9):10.1007/s13361-012-0419-6.
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is an established method for the interrogation of protein conformation and dynamics. While the data analysis challenge of HDX-MS has been addressed by a number of software packages, new computational tools are needed to keep pace with the improved methods and throughput of this technique. To address these needs, we report an integrated desktop program titled HDX Workbench, which facilitates automation, management, visualization, and statistical cross-comparison of large HDX data sets. Using the software, validated data analysis can be achieved at the rate of generation. The application is available at the project home page
PMCID: PMC3808162  PMID: 22692830
Mass spectrometry; HDX; Software; H/D exchange; Protein dynamics; Automation
6.  Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases 
Science (New York, N.Y.)  2011;335(6064):85-88.
Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.
PMCID: PMC3584687  PMID: 22116026
7.  Ligand-dependent perturbation of the conformational ensemble for the GPCR beta2 adrenergic receptor revealed by HDX 
Structure (London, England : 1993)  2011;19(10):1424-1432.
Mechanism of G-protein coupled receptor (GPCR) activation and their modulation by functionally distinct ligands remains elusive. Using the technique of amide hydrogen/deuterium exchange coupled with mass spectrometry we examined the ligand-induced changes in conformational states and stability within the beta-2-adrenergic receptor (β2AR). Differential HDX reveals ligand-specific alterations in the energy landscape of the receptor’s conformational ensemble. The inverse agonists timolol and carazolol were found to be most stabilizing even compared to the antagonist alprenolol, notably in intracellular regions where G-proteins are proposed to bind, while the agonist isoproterenol induced the largest degree of conformational mobility. The partial agonist clenbuterol displayed found in both the inverse agonists and the agonist. This study confirms the regional plasticity of the receptor, supports current models for GPCR signaling, and characterizes unique conformations spanning the entire receptor sequence stabilized solely by functionally selective ligands all of which differ from the apo state of the receptor.
PMCID: PMC3196059  PMID: 21889352
GPCRs; activation mechanism; conformational stability; conformational ensemble; functional selectivity; ligand-biased signaling
8.  Stable Isotope Labeling Strategy for Protein–Ligand Binding Analysis in Multi-Component Protein Mixtures 
Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein–ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein–ligand binding interactions. The protocol utilizes an H216O2 and H218O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein–ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique’s ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein–protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).
PMCID: PMC3085011  PMID: 21472561
Protein folding; Ligand binding; Thermodynamics; Covalent labeling; Quantitative proteomics; Cyclophilin A; Cyclosporin A; Calcineurin
9.  Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions 
Expert review of proteomics  2011;8(1):43-59.
Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery.
PMCID: PMC3113475  PMID: 21329427
GPCR; HDX; ligand; MS; nuclear receptor; screening
10.  Discovery of Novel Cyclophilin A Ligands Using an H/D Exchange- and Mass Spectrometry-Based Strategy 
Journal of biomolecular screening  2010;15(9):1051-1062.
Cyclophilin A (CypA) is an overexpressed protein in lung cancer tumors and as a result is a potential therapeutic and diagnostic target. Here we utilize an H/D exchange- and MALDI mass spectrometry-based assay, termed single-point SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange), to screen two chemical libraries, including the 1280-compound LOPAC library and the 9600 compound DIVERSet library, for binding to CypA. This work represents the first application of single-point SUPREX using a pooled ligand approach, which we demonstrate is capable of screening rates as fast as six seconds/ligand. The false positive and false negative rates determined in the current work using a set of control samples were 0% and 9%, respectively. A false positive rate of 20% was found in screening the actual libraries. Eight novel ligands to CypA were discovered including: 2-(α-naphthoyl)ethyltrimethyl-ammonium iodide, (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile, 3-(N-benzyl-N-isopropyl)amino-1-(naphthalen-2-yl)propan-1-one, cis-diammineplatinum (II) chloride, 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione, N-(3-chloro-1,4-dioxo-1,4-dihydro-2-naphthalenyl)-N-cyclohexylacetamide, 1-[2-(3,4-dimethoxyphenyl)ethyl]-1H-pyrrole-2,5-dione, and 4-(2-methoxy-4-nitrophenyl)-1-methyl-10-oxa-4-azatricyclo[,6~]dec-8-ene-3,5-dione. These compounds, which had moderate binding affinities to CypA (i.e., Kd values in the low micromolar range), provide new molecular scaffolds that might be useful in the development of CypA targeted diagnostic imaging or therapeutic agents for lung cancer.
PMCID: PMC3197229  PMID: 20855564
Cyclophilin A; Matrix-Assisted Laser Desorption/Ionization; amide H/D exchange; high-throughput screening

Results 1-10 (10)