Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen

Beaudet, Lucille; Bédard, Julie; Breton, Billy; Mercuri, Roberto J.; Budarf, Marcia L.

doi:10.1101/gr.172501

Cited by 58 publications

(32 citation statements)

References 43 publications

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“…Binding was measured by flowing 370 nM FabG in 10 mM HEPES, 150 mM NaCl, pH 7. FabG-ACP Binding Measured by AlphaScreen-AlphaScreen technology is an approach to study biological interactions by energy transfer (22). Upon laser excitation, a chemical signal is generated on the donor beads (streptavidin-coated).…”

Section: Materials-amershammentioning

confidence: 99%

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

133

148

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We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on ␤-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix ␣2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix ␣2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.Fatty acid biosynthesis in bacteria and plants is carried out by a series of enzymes that are collectively known as the type II fatty-acid synthase and is most extensively studied in the Escherichia coli system (1, 2). ACP 1 is a small, acidic protein that functions as the central acyl group carrier in type II fatty-acid synthase systems. The ACPs are rod-shaped proteins with a preponderance of acidic residues organized into four ␣-helices (3-7). The acyl intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group (8), which is attached via a phosphodiester linkage to Ser-36 located at the beginning of the second helical segment. The primary gene product is an apoprotein that is converted to ACP by the transfer of the 4Ј-phosphopantetheine moiety of CoA to Ser-36 by [ACP]synthase (AcpS) (9, 10). ACP performs two functions. First, it sequesters the growing acyl chain from the aqueous environment, and second, upon binding to one of the type II proteins, it releases its grip on the fatty acid, which is inserted into the active site cavity of the enzyme. ACPs are widely distributed in nature and are easily recognized by their significant primary sequence identity, particularly at the prosthetic group attachment site and extending along helix ␣2 (11). These similarities are thought to underlie the observation that ACPs from virtually any source are substrates for AcpS and function with the fatty acid biosynthetic enzymes of E. coli (11). However, the individual enzymes of type II fatty acid synthesis do not share a primary structure motif that would define the presence of a common ACP-binding motif.The molecular details that govern the specific interactions between ACP and the type II enzymes are poorly known. The analysis of the binding of ACP to FabH points to ionic interactions playing a determinant ...

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Section: Materials-amershammentioning

confidence: 99%

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Zhang¹,

Zheng

et al. 2003

Journal of Biological Chemistry

133

148

View full text Add to dashboard Cite

show abstract

“…SpFabF-ACP Interactions-AlphaScreen technology is an experimental method to quantitate protein-protein interactions (33). Upon laser excitation, a chemical signal is generated on the donor beads (streptavidin-coated).…”

mentioning

confidence: 99%

Roles of the Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae

Zhang

Hurlbert

White

et al. 2006

Journal of Biological Chemistry

107

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␤-Ketoacyl-ACP synthases catalyze the condensation steps in fatty acid and polyketide synthesis and are targets for the development of novel antibiotics and anti-obesity and anti-cancer agents. The roles of the active site residues in Streptococcus pneumoniae FabF (␤-ketoacyl-ACP synthase II; SpFabF) were investigated to clarify the mechanism for this enzyme superfamily. The nucleophilic cysteine of the active site triad was required for acyl-enzyme formation and the overall condensation activity. functions as a gatekeeper that controls the order of substrate addition. These data assign specific roles for each active site residue and lead to a revised general mechanism for this important class of enzymes.The condensing enzymes play a central role in fatty acid biosynthesis by elongating the growing acyl chain by two carbon atoms to initiate each elongation cycle (1, 2). Somewhat uniquely in biological synthesis, the enzymes create a carbon-carbon bond via a Claisen-like condensation reaction (3). Specifically, they catalyze the condensation of malonyl-acyl carrier protein (ACP) 2 with an acyl-ACP intermediate via a two-step ping-pong kinetic mechanism. In the first step, an acyl chain from either acyl-CoA or acyl-ACP is transferred to an active site cysteine, and the cofactor is released. During the second step, malonyl-ACP binds, and a carbanion is generated on the C2 of malonate concomitant with the release of the C3 carboxyl group (4 -6). The carbanion then attacks the acyl-enzyme intermediate to produce the ␤-ketoacyl-ACP product. In the dissociated, type II synthases, the condensation reaction is carried out by monofunctional enzymes (7), and most bacteria have only a single elongation-condensing enzyme that belongs to the FabF class. In mammals and yeast, the condensing enzyme component, KS, is fused into a multidomain complex referred to as the type I or associated FAS system (8). However, it is clear from primary sequence analysis that the active site of the FAS I condensation module is very similar to the FAS II elongation enzymes (Fig. 1A). The polyketide synthases also contain a condensing enzyme module in which the same signature active site residues can be identified (Fig. 1A).The importance of the elongation condensing enzymes in regulating fatty acid formation (7, 9) and the unique chemistry of the reaction that they catalyze (3) have focused our interest on understanding the specific tasks of each active site residue in catalysis. In addition, these enzymes have emerged as attractive targets for the development of new broadspectrum antibiotics (10 -12) and anti-obesity/anti-cancer drugs (13-17), and there is growing interest in engineering the polyketide synthases to produce novel therapeutic agents (18). These efforts will be facilitated by a complete mechanistic understanding of the active site. At their catalytic cores, the elongation enzymes possess a Cys-His-His triad. These residues have been mutated and are thought to be critical to the overall forward condensation reaction (8, 19 -22), al...

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“…Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen TM (9) and FlashPlate® (10). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners.…”

mentioning

confidence: 99%

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

Caruso

Jenna

Beaulne³

et al. 2005

Molecular & Cellular Proteomics

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To date phylogeny has been used to compare entire families of proteins based on their nucleotide or amino acid sequence. Here we developed a novel analytical platform allowing a systematic comparison of protein families based on their biochemical properties. This approach was validated on the Rho subfamily of GTPases. We used two high throughput methods, referred to as AlphaScreen Ras GTPases are small GTP-binding proteins involved in diverse cellular processes such as apoptosis, cell proliferation/differentiation, cytoskeleton reorganization, and membrane trafficking. These proteins cycle between an inactive GDP-bound form and an active GTP-bound form. Under their GTP-bound form their active conformation allows them to interact with effector molecules and to generate specific biological responses (1). Two intrinsic enzymatic activities regulate the balance between GTP-bound and GDP-bound conformations: 1) GDP/GTP exchange and 2) GTP hydrolysis activities (2). Whereas the intrinsic enzymatic activities have been characterized for a few of these proteins, such studies were carried out in non-systematic manners and by different research groups (3-6).The analysis of full genome sequences has allowed large scale biology experimental approaches consisting of systematic characterization of entire families of proteins instead of investigating them individually. Developing such "functional proteomic" approaches requires the design of high throughput and versatile experimental procedures (7,8). For the study of small G proteins, conventional filtration assays, although powerful at small scale, are not appropriate for systematic studies. Indeed this methodology has a low to very low throughput, is not versatile, and requires large amounts of reagents. To overcome these issues and characterize GTP-binding proteins of the Ras superfamily in a systematic manner, we developed a robust assay platform using two well adopted high throughput technologies, AlphaScreen TM (9) and FlashPlate® (10). AlphaScreen is a bead-based non-radioactive and homogenous proximity assay used to measure interaction between biological binding partners. The principle of this technology relies on the use of a Donor bead and an Acceptor bead that generate a light signal when brought into proximity (Ͻ200 nm). Upon laser excitation at 680 nm, the Donor beads, containing a photosensitizer, will generate short lived singlet oxygen that can diffuse only a short distance before returning to the ground state. The Acceptor beads, containing chemiluminescers and fluorophores, will react with this singlet oxygen and will emit an amplified light signal measurable at ϳ600 nm. AlphaScreen (PerkinElmer) provides highly versatile, sensitive, and homogeneous assays that allow us to perform studies at a higher throughput and at a lower cost.FlashPlates are white polystyrene microplates in which the interior of wells is coated with a thin layer of polystyrenebased scintillation reagent. The principle of this homogeneous radiometric technology is based on proximity betwe...

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Homogeneous Assays for Single-Nucleotide Polymorphism Typing Using AlphaScreen

Cited by 58 publications

References 43 publications

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Key Residues Responsible for Acyl Carrier Protein and β-Ketoacyl-Acyl Carrier Protein Reductase (FabG) Interaction

Roles of the Active Site Water, Histidine 303, and Phenylalanine 396 in the Catalytic Mechanism of the Elongation Condensing Enzyme of Streptococcus pneumoniae

Biochemical Clustering of Monomeric GTPases of the Ras Superfamily

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