Chromatographic Methods to Study Protein–Protein Interactions

Beeckmans, Sonia

doi:10.1006/meth.1999.0857

Cited by 52 publications

(36 citation statements)

References 127 publications

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“…For this purpose a defined amount of the tested interaction partner (GluTR) is injected onto a gel filtration system equilibrated with GSA-AM, and elution profiles are recorded spectroscopically. The binding of GluTR to GSA-AM results in a local deficit of GSA-AM in the eluent, allowing the determination of the binding constant (15,16). An analytical Superdex 200 PC 3.2/30 (Amersham Biosciences) was used to investigate the binding of GluTR to GSA-AM.…”

Section: Methodsmentioning

confidence: 99%

Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis

Lüer

Schauer

Möbius

et al. 2005

Journal of Biological Chemistry

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In Escherichia coli the first common precursor of all tetrapyrroles, 5-aminolevulinic acid, is synthesized from glutamyl-tRNA (Glu-tRNA Glu ) in a two-step reaction catalyzed by glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM). To protect the highly reactive reaction intermediate glutamate-1-semialdehyde (GSA), a tight complex between these two enzymes was proposed based on their solved crystal structures. The existence of this hypothetical complex was verified by two independent biochemical techniques. Co-immunoprecipitation experiments using antibodies directed against E. coli GluTR and GSA-AM demonstrated the physical interaction of both enzymes in E. coli cell-free extracts and between the recombinant purified enzymes. Additionally, the formation of a GluTR⅐GSA-AM complex was identified by gel permeation chromatography. Complex formation was found independent of Glu-tRNA Glu and cofactors. The analysis of a GluTR mutant truncated in the 80-amino acid C-terminal dimerization domain (GluTR-A338Stop) revealed the importance of GluTR dimerization for complex formation. The in silico model of the E. coli GluTR⅐GSA-AM complex suggested direct metabolic channeling between both enzymes to protect the reactive aldehyde species GSA. In accordance with this proposal, side product formation catalyzed by GluTR was observed via high performance liquid chromatography analysis in the absence of the GluTR⅐GSA-AM complex.In plants, green algae, archaea, and most bacteria the common precursor molecule of all tetrapyrroles, 5-aminolevulinic acid (ALA), 1 is synthesized from tRNA-bound glutamate (GlutRNA Glu ) in a two-step reaction (1, 2). In the first step, the NADPH-dependent glutamyl-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA to glutamate-1-semialdehyde (GSA). In a subsequent transamination reaction this aldehyde is transformed into ALA by the pyridoxal 5Ј-phosphatedependent enzyme glutamate-1-semialdehyde 2,1-aminomutase (GSA-AM) (3).Recently the catalytic mechanism of GluTR and its structural basis have been elucidated in a combined biochemical and structural investigation using the recombinant enzyme from the extreme thermophilic archaean Methanopyrus kandleri (4, 5). The crystal structure of GluTR reveals an unusual extended V-shaped dimer with each monomer consisting of three distinct domains arranged along a curved "spinal" ␣-helix. The N-terminal catalytic domain specifically recognizes the glutamate moiety of the substrate. The active site was identified by cocrystallization of the competitive inhibitor glutamycin representing the 3Ј-terminal end of the natural substrate. During catalysis a nucleophilic cysteine residue attacks the aminoacyl linkage of the glutamate to its cognate tRNA and generates an enzyme-bound thioester intermediate. This intermediate has been biochemically trapped and visualized. For this purpose a new purification strategy for the Escherichia coli enzyme has been developed (6, 7). The thioester intermediate gets finally reduced by direc...

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Section: Methodsmentioning

confidence: 99%

Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis

Lüer

Schauer

Möbius

et al. 2005

Journal of Biological Chemistry

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“…Although this is valuable information in itself and reduces the problem space by several orders of magnitude, it is still necessary to validate the putative protein interactions. Once the target proteins are identified, their binding partners can be found easily using conventional techniques such as affinity chromatography (12) or two-hybrid analysis. To provide a complete understanding of the interaction, it also necessary to confirm that the putative interaction occurs using some alternative method.…”

Section: Resultsmentioning

confidence: 99%

“…A number of techniques have been used to study individual protein-protein interactions, including protein cross-linking (1-3), green fluorescent protein (4,5), phage display (6, 7), the two-hybrid system (8), protein arrays (9), fiber optic evanescent wave sensors (10,11), chromatographic techniques (12), and fluorescence resonance energy transfer (13,14). However, these methods are generally only useful for screening one bait protein at a time.…”

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confidence: 99%

Isolation of Protein Subpopulations Undergoing Protein-Protein Interactions

Nelson

Backlund

Yergey

et al. 2002

Molecular & Cellular Proteomics

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A new method is described for isolating and identifying proteins participating in protein-protein interactions in a complex mixture. The method uses a cyanogen bromideactivated Sepharose matrix to isolate proteins that are non-covalently bound to other proteins. Because the proteins are accessible to chemical manipulation, mass spectrometric identification of the proteins can yield information on specific classes of interacting proteins, such as calcium-dependent or substrate-dependent protein interactions. This permits selection of a subpopulation of proteins from a complex mixture on the basis of specified interaction criteria. The new method has the advantage of screening the entire proteome simultaneously, unlike the two-hybrid system or phage display, which can only detect proteins binding to a single bait protein at a time. The method was tested by selecting rat brain extract for proteins exhibiting calcium-dependent protein interactions. Of 12 proteins identified by mass spectrometry, eight were either known calcium-binding proteins or proteins with known calcium-dependent protein interactions, indicating that the method is capable of enriching a subpopulation of proteins from a complex mixture on the basis of a specific class of protein interactions. Because only naturally occurring interactions of proteins in their native state are observed, this method will have wide applicability to studies of protein interactions in tissue samples and autopsy specimens, for screening for perturbations of protein-protein interactions by signaling molecules, pharmacological agents or toxins, and screening for differences between cancerous and untransformed cells.

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“…With histidine-tagged forms of MDH2 and PCK1, it was possible to purify sufficient quantities of both enzymes to apply the classical Hummel-Dreyer chromatography method for binding analyses (37)(38)(39). For this, we developed a gel filtration system for resolution of MDH2 and PCK1 and determined their elution patterns relative to a set of standard proteins (inset, Fig.…”

Section: Differential Function Of Mdh2 and ⌬Nmdh2mentioning

confidence: 99%

Physical and Genetic Interactions of Cytosolic Malate Dehydrogenase with Other Gluconeogenic Enzymes

Gibson

McAlister-Henn

2003

Journal of Biological Chemistry

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A truncated form (⌬nMDH2) of yeast cytosolic malate dehydrogenase (MDH2) lacking 12 residues on the amino terminus was found to be inadequate for gluconeogenic function in vivo because the mutant enzyme fails to restore growth of a ⌬mdh2 strain on minimal medium with ethanol or acetate as the carbon source. The ⌬nMDH2 enzyme was also previously found to be refractory to the rapid glucose-induced inactivation and degradation observed for authentic MDH2. In contrast, kinetic properties measured for purified forms of MDH2 and ⌬nMDH2 enzymes are very similar. Yeast two-hybrid assays indicate weak interactions between MDH2 and yeast phosphoenolpyruvate carboxykinase (PCK1) and between MDH2 and fructose-1,6-bisphosphatase (FBP1). These interactions are not observed for ⌬nMDH2, suggesting that differences in cellular function between authentic and truncated forms of MDH2 may be related to their ability to interact with other gluconeogenic enzymes. Additional evidence was obtained for interaction of MDH2 with PCK1 using Hummel-Dreyer gel filtration chromatography, and for interactions of MDH2 with PCK1 and with FBP1 using surface plasmon resonance. Experiments with the latter technique demonstrated a much lower affinity for interaction of ⌬nMDH2 with PCK1 and no interaction between ⌬nMDH2 and FBP1. These results suggest that the interactions of MDH2 with other gluconeogenic enzymes are dependent on the amino terminus of the enzyme, and that these interactions are important for gluconeogenic function in vivo.Three differentially compartmentalized isozymes of malate dehydrogenase (MDH) 1 in Saccharomyces cerevisiae catalyze the NAD(H)-specific interconversion of malate and oxaloacetate. Mitochondrial MDH1 catalyzes a reaction of the tricarboxylic acid cycle, and disruption of the corresponding gene results in an inability to grow with acetate as a carbon source (1, 2), a phenotype shared with yeast mutants containing disruptions in genes encoding other tricarboxylic acid cycle enzymes (3-5). Cytosolic MDH2 is a gluconeogenic enzyme and is required for growth on minimal medium with ethanol or acetate as the carbon source (6), as are the other yeast gluconeogenic enzymes phosphoenolpyruvate carboxykinase (PCK1) (7) and fructose-1,6-bisphosphatase (FBP1) (8). Peroxisomal MDH3 is proposed to catalyze a step in the glyoxylate pathway, which permits formation of C 4 metabolites from C 2 precursors (9, 10), and has also been shown to be necessary for growth with oleate as the carbon source (11), suggesting an additional role in providing NADH for peroxisomal ␤-oxidation.The yeast MDH isozymes are all homodimers with similar subunit M r values (33,500 for MDH1; 40,700 for MDH2; and 37,200 for MDH3), and they share primary sequence identities of 43 to 50%. Distinct differences among the enzymes include a 17-residue mitochondrial targeting sequence for MDH1 that is removed upon import (2), and a carboxyl-terminal tripeptide targeting sequence (Ser-Lys-Leu) required for peroxisomal import of MDH3 (11). In addition, MDH2 has a 12-r...

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Chromatographic Methods to Study Protein–Protein Interactions

Cited by 52 publications

References 127 publications

Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis

Complex Formation between Glutamyl-tRNA Reductase and Glutamate-1-semialdehyde 2,1-Aminomutase in Escherichia coli during the Initial Reactions of Porphyrin Biosynthesis

Isolation of Protein Subpopulations Undergoing Protein-Protein Interactions

Physical and Genetic Interactions of Cytosolic Malate Dehydrogenase with Other Gluconeogenic Enzymes

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