Reactivity of histidine and lysine side-chains with diethylpyrocarbonate — A method to identify surface exposed residues in proteins

Hnı́zda, Aleš; Šantrůček, Jiří; Šanda, Miloslav; Strohalm, Martin; Kodı́ček, Milan

doi:10.1016/j.jbbm.2007.07.004

Cited by 25 publications

(23 citation statements)

References 23 publications

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“…Furthermore, the mutagenesis revealed that the hydroxyl group of T34 is important because the residue could be replaced by Ser without loss of activity. The observation that LpxR was fully inhibited by diethylpyrocarbonate proved that a histidine, presumably H122, is essential for catalysis (11). Furthermore, we found that dissolved imidazole (100 mM) restored the catalytically inactive H122A mutant protein to Ϸ30% of the wild-type activity by complementing the absent imidazole ring.…”

Section: Resultsmentioning

confidence: 51%

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Rutten¹,

Mannie²,

Stead³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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The lipid A portion of lipopolysaccharide, the major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is toxic to humans. Modification of lipid A by enzymes often reduces its toxicity. The outer-membrane protein LpxR from Salmonella typhimurium is a lipid A-modifying enzyme. It removes the 3-acyloxyacyl moiety of the lipid A portion of lipopolysaccharide in a Ca 2؉ -dependent manner. Here, we present the crystal structure of S. typhimurium LpxR, crystallized in the presence of zinc ions. The structure, a 12-stranded ␤-barrel, reveals that the active site is located between the barrel wall and an ␣-helix formed by an extracellular loop. Based on site-directed mutagenesis and modeling of a substrate on the active site, we propose a catalytic mechanism similar to that of phospholipase A2, in which a Ca 2؉ forms the oxyanion hole and a histidine activates a water molecule (or a cascade of two water molecules) that subsequently attacks the carbonyl oxygen of the scissile bond.crystal structure ͉ endotoxin ͉ lipopolysaccharide ͉ outer-membrane protein ͉ phospholipase

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Section: Resultsmentioning

confidence: 51%

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Rutten¹,

Mannie²,

Stead³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…Another approach is the chemical modification of the deprotonated ring nitrogen of His with diethyl pyrocarbonate (DEPC) that can be followed by UV spectroscopy or mass spectrometry [17–19]. The method is widely used for the identification of surface-exposed residues or to tackle the His protonation state [18]. More recently, DEPC was applied to differentiate between metal ion coordinated and free His in MTs [20].…”

Section: Introductionmentioning

confidence: 99%

“…concomitant modification of the protein N-terminus as well as lysine and tyrosine residues [18]. At increased DEPC-to-protein ratios additional adducts can appear in the mass spectra resulting from modification of both His imidazole nitrogen atoms with DEPC (+ 162 Da) and hydrolysis of the latter (+ 134 Da) [18]. In addition, interpretation of the results can become challenging as incomplete or failed modification can originate from multiple factors.…”

Section: Introductionmentioning

confidence: 99%

Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata

et al. 2017

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The superfamily of metallothioneins (MTs) combines a diverse group of metalloproteins, sharing the characteristics of rather low molecular weight and high cysteine content. The latter provides MTs with the capability to coordinate thiophilic metal ions, in particular those with a d10 electron configuration. The sub-family of plant MT3 proteins is only poorly characterized and there is a complete lack of three-dimensional structure information. Building upon our previous results on the Musa acuminata MT3 (musMT3) protein, the focus of the present work is to understand the metal cluster formation process, the role of the single histidine residue present in musMT3, and the metal ion binding affinity. We concentrate our efforts on the coordination of ZnII and CdII ions, using CoII as a spectroscopic probe for ZnII binding. The overall protein-fold is analysed with a combination of limited proteolytic digestion, mass spectrometry, and dynamic light scattering. Histidine coordination of metal ions is probed with extended X-ray absorption fine structure spectroscopy and CoII titration experiments. Initial experiments with isothermal titration calorimetry provide insights into the thermodynamics of metal ion binding.

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“…Our results indicate that DEPC prevents the direct binding of Spa to anti-LukS-PV. In addition to histidine, lysine, tyrosine, serine and threonine residues can be modified by DEPC (Hnizda et al, 2008; Mendoza and Vachet, 2008). Because these residues may be found in the antigen-binding site of antibodies, we determined if the presence of DEPC interferes with the binding of LukS-PV to anti-LukS-PV in the sandwich ELISA.…”

Section: Resultsmentioning

confidence: 99%

Detection and quantification of Panton–Valentine leukocidin in Staphylococcus aureus cultures by ELISA and Western blotting: Diethylpyrocarbonate inhibits binding of protein A to IgG

Nguyen

Rocha

Chintalacharuvu

et al. 2010

Journal of Immunological Methods

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Enzyme-linked immunosorbent assay (ELISA) and Western blotting are common techniques used to detect and quantify proteins in Staphylococcus aureus culture supernatants, such as PantonValentine leukocidin (PVL). However, protein A (Spa) secreted by most S. aureus strains may interfere with these assays by binding to the capturing and detecting antibodies. Here, we have shown that the addition of diethylpyrocarbonate (DEPC) inhibits the binding of Spa to rabbit anti-PVL used as the capturing antibody in ELISA. In Western blotting, the presence of DEPC prevented the binding of detecting antibody to Spa. These modified ELISA and Western blot techniques should prove useful for detecting and quantifying proteins in S. aureus cultures supernatants.

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Reactivity of histidine and lysine side-chains with diethylpyrocarbonate — A method to identify surface exposed residues in proteins

Cited by 25 publications

References 23 publications

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Active-site architecture and catalytic mechanism of the lipid A deacylase LpxR of Salmonella typhimurium

Further insights into the metal ion binding abilities and the metalation pathway of a plant metallothionein from Musa acuminata

Detection and quantification of Panton–Valentine leukocidin in Staphylococcus aureus cultures by ELISA and Western blotting: Diethylpyrocarbonate inhibits binding of protein A to IgG

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