Publications by authors named "Robert H H van den Heuvel"

37 Publications

Cofactor binding protects flavodoxin against oxidative stress.

PLoS One 2012 19;7(7):e41363. Epub 2012 Jul 19.

Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.

In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modification.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041363PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400614PMC
December 2012

Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS.

Nucleic Acids Res 2011 Oct 6;39(18):8052-64. Epub 2011 Jul 6.

Biomolecular Mass Spectrometry and Proteomics Group, and Center for Biomedical Genetics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.

The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.
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http://dx.doi.org/10.1093/nar/gkr498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3185415PMC
October 2011

Structure of the ribosome associating GTPase HflX.

Proteins 2010 Feb;78(3):705-13

Laboratory of Microbiology, Wageningen University, Wageningen, The Netherlands.

The HflX-family is a widely distributed but poorly characterized family of translation factor-related guanosine triphosphatases (GTPases) that interact with the large ribosomal subunit. This study describes the crystal structure of HflX from Sulfolobus solfataricus solved to 2.0-A resolution in apo- and GDP-bound forms. The enzyme displays a two-domain architecture with a novel "HflX domain" at the N-terminus, and a classical G-domain at the C-terminus. The HflX domain is composed of a four-stranded parallel beta-sheet flanked by two alpha-helices on either side, and an anti-parallel coiled coil of two long alpha-helices that lead to the G-domain. The cleft between the two domains accommodates the nucleotide binding site as well as the switch II region, which mediates interactions between the two domains. Conformational changes of the switch regions are therefore anticipated to reposition the HflX-domain upon GTP-binding. Slow GTPase activity has been confirmed, with an HflX domain deletion mutant exhibiting a 24-fold enhanced turnover rate, suggesting a regulatory role for the HflX domain. The conserved positively charged surface patches of the HflX-domain may mediate interaction with the large ribosomal subunit. The present study provides a structural basis to uncover the functional role of this GTPases family whose function is largely unknown.
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http://dx.doi.org/10.1002/prot.22599DOI Listing
February 2010

Microsomal glutathione transferase 1 exhibits one-third-of-the-sites-reactivity towards glutathione.

Arch Biochem Biophys 2009 Jul 3;487(1):42-8. Epub 2009 May 3.

Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden.

The trimeric membrane protein microsomal glutathione transferase 1 (MGST1) possesses glutathione transferase and peroxidase activity. Previous data indicated one active site/trimer whereas structural data suggests three GSH-binding sites. Here we have determined ligand interactions of MGST1 by several techniques. Nanoelectrospray mass spectrometry of native MGST1 revealed binding of three GSH molecules/trimer and equilibrium dialysis showed three product molecules/trimer (K(d)=320+/-50 microM). All three product molecules could be competed out with GSH. Reinvestigation of GSH-binding showed one high affinity site per trimer, consistent with earlier data. Using single turnover stopped flow kinetic measurements, K(d) could be determined for a low affinity GSH-binding site (2.5+/-0.5 mM). Thus we can reconcile previous observations and show here that MGST1 contains three active sites with different affinities for GSH and that only the high affinity site is catalytically competent.
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http://dx.doi.org/10.1016/j.abb.2009.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2996728PMC
July 2009

Crystal structure and biochemical properties of a novel thermostable esterase containing an immunoglobulin-like domain.

J Mol Biol 2009 Jan 5;385(3):949-62. Epub 2008 Nov 5.

Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands.

Comparative analysis of the genome of the hyperthermophilic bacterium Thermotoga maritima revealed a hypothetical protein (EstA) with typical esterase features. The EstA protein was functionally produced in Escherichia coli and purified to homogeneity. It indeed displayed esterase activity with optima at or above 95 degrees C and at pH 8.5, with a preference for esters with short acyl chains (C2-C10). Its 2.6-A-resolution crystal structure revealed a classical alpha/beta hydrolase domain with a catalytic triad consisting of a serine, an aspartate, and a histidine. EstA is irreversibly inhibited by the organophosphate paraoxon. A 3.0-A-resolution structure confirmed that this inhibitor binds covalently to the catalytic serine residue of EstA. Remarkably, the structure also revealed the presence of an N-terminal immunoglobulin (Ig)-like domain, which is unprecedented among esterases. EstA forms a hexamer both in the crystal and in solution. Electron microscopy showed that the hexamer in solution is identical with the hexamer in the crystal, which is formed by two trimers, with the N-terminal domains facing each other. Mutational studies confirmed that residues Phe89, Phe112, Phe116, Phe246, and Trp377 affect enzyme activity. A truncated mutant of EstA, in which the Ig-like domain was removed, showed only 5% of wild-type activity, had lower thermostability, and failed to form hexamers. These data suggest that the Ig-like domain plays an important role in the enzyme multimerization and activity of EstA.
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http://dx.doi.org/10.1016/j.jmb.2008.10.075DOI Listing
January 2009

Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process.

FEBS J 2008 Oct 11;275(20):5191-200. Epub 2008 Sep 11.

Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD )= 2.3 microM, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06649.xDOI Listing
October 2008

ADP competes with FAD binding in putrescine oxidase.

J Biol Chem 2008 Oct 4;283(42):28259-64. Epub 2008 Aug 4.

Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.
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http://dx.doi.org/10.1074/jbc.M803255200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661395PMC
October 2008

Hydroquinone dioxygenase from pseudomonas fluorescens ACB: a novel member of the family of nonheme-iron(II)-dependent dioxygenases.

J Bacteriol 2008 Aug 23;190(15):5199-209. Epub 2008 May 23.

Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands.

Hydroquinone 1,2-dioxygenase (HQDO), an enzyme involved in the catabolism of 4-hydroxyacetophenone in Pseudomonas fluorescens ACB, was purified to apparent homogeneity. Ligandation with 4-hydroxybenzoate prevented the enzyme from irreversible inactivation. HQDO was activated by iron(II) ions and catalyzed the ring fission of a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes. HQDO was inactivated by 2,2'-dipyridyl, o-phenanthroline, and hydrogen peroxide and inhibited by phenolic compounds. The inhibition with 4-hydroxybenzoate (K(i) = 14 microM) was competitive with hydroquinone. Online size-exclusion chromatography-mass spectrometry revealed that HQDO is an alpha2beta2 heterotetramer of 112.4 kDa, which is composed of an alpha-subunit of 17.8 kDa and a beta-subunit of 38.3 kDa. Each beta-subunit binds one molecule of 4-hydroxybenzoate and one iron(II) ion. N-terminal sequencing and peptide mapping and sequencing based on matrix-assisted laser desorption ionization--two-stage time of flight analysis established that the HQDO subunits are encoded by neighboring open reading frames (hapC and hapD) of a gene cluster, implicated to be involved in 4-hydroxyacetophenone degradation. HQDO is a novel member of the family of nonheme-iron(II)-dependent dioxygenases. The enzyme shows insignificant sequence identity with known dioxygenases.
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http://dx.doi.org/10.1128/JB.01945-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2493252PMC
August 2008

Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1.

FEBS J 2007 May 5;274(9):2311-21. Epub 2007 Apr 5.

Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.

A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05767.xDOI Listing
May 2007

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

Nucleic Acids Res 2007 21;35(5):1737-49. Epub 2007 Feb 21.

Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Department of Biomolecular Mass Spectrometry, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.
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http://dx.doi.org/10.1093/nar/gkm073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865072PMC
April 2007

Structure and function of bacterial kid-kis and related toxin-antitoxin systems.

Protein Pept Lett 2007 ;14(2):113-24

Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Toxin-antitoxin systems were discovered as plasmid auxiliary maintenance cassettes. In recent years, an increasing amount of structural and functional information has become available about the proteins involved, allowing the understanding of bacterial cell growth inhibition by the toxins on a molecular level. A well-studied TA system is formed by the proteins Kid and Kis, encoded by the parD operon of the Escherichia coli plasmid R1. The toxicity of Kid has been related to its endoribonuclease activity, which is counteracted by binding of the antitoxin Kis at the proposed active site. In this review, the structural studies on the Kid-Kis system are compared to those of three related toxin-antitoxin systems: MazF-MazE, CcdB-CcdA and RelE-RelB.
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http://dx.doi.org/10.2174/092986607779816096DOI Listing
June 2007

Transcriptional activation by CprK1 is regulated by protein structural changes induced by effector binding and redox state.

J Biol Chem 2007 Apr 15;282(15):11281-90. Epub 2007 Feb 15.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

The transcriptional activator CprK1 from Desulfitobacterium-hafniense, a member of the ubiquitous cAMP receptor protein/fumarate nitrate reduction regulatory protein family, activates transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. 3-chloro-4-hydroxyphenylacetate is a known effector for CprK1, which interacts tightly with the protein, and induces binding to a specific DNA sequence ("dehalobox," TTAAT--ATTAA) located in the promoter region of chlorophenol reductive dehalogenase genes. Despite the availability of recent x-ray structures of two CprK proteins in distinct states, the mechanism by which CprK1 activates transcription is poorly understood. In the present study, we have investigated the mechanism of CprK1 activation and its effector specificity. By using macromolecular native mass spectrometry and DNA binding assays, analogues of 3-chloro-4-hydroxyphenylacetate that have a halogenated group at the ortho position and a chloride or acetic acid group at the para position were found to be potent effectors for CprK1. By using limited proteolysis it was demonstrated that CprK1 requires a cascade of structural events to interact with dehalobox dsDNA. Upon reduction of the intermolecular disulfide bridge in oxidized CprK1, the protein becomes more dynamic, but this alone is not sufficient for DNA binding. Activation of CprK1 is a typical example of allosteric regulation; the binding of a potent effector molecule to reduced CprK1 induces local changes in the N-terminal effector binding domain, which subsequently may lead to changes in the hinge region and as such to structural changes in the DNA binding domain that are required for specific DNA binding.
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http://dx.doi.org/10.1074/jbc.M611177200DOI Listing
April 2007

Surface-plasmon-resonance-based chemical proteomics: efficient specific extraction and semiquantitative identification of cyclic nucleotide-binding proteins from cellular lysates by using a combination of surface plasmon resonance, sequential elution and liquid chromatography-tandem mass spectrometry.

Chembiochem 2007 Feb;8(3):298-305

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

Chemical proteomics is a powerful methodology for identifying the cellular targets of small molecules, however, it is biased towards abundant proteins. Therefore, quantitative strategies are needed to distinguish between specific and nonspecific interactions. Here, we explore the potential of the combination of surface plasmon resonance (SPR) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) as an alternative approach in chemical proteomics. We coupled cGMP molecules to the SPR chip, and monitored the binding and dissociation of proteins from a human lysate by using sequential elution steps and SPR. The eluted proteins were subsequently identified by LC-MS/MS. Our approach enabled the efficient and selective extraction of low-abundant cyclic-nucleotide-binding proteins such as cGMP-dependent protein kinase, and a quantitative assessment of the less- and nonspecific competitive binding proteins. The data show that SPR-based chemical proteomics is a promising alternative for the efficient specific extraction and quantitative identification of small-molecule-binding proteins from complex mixtures.
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http://dx.doi.org/10.1002/cbic.200600449DOI Listing
February 2007

Interactions between the toxin Kid of the bacterial parD system and the antitoxins Kis and MazE.

Proteins 2007 Apr;67(1):219-31

Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Utrecht, The Netherlands.

The proteins Kid and Kis are the toxin and antitoxin, respectively, encoded by the parD operon of Escherichia coli plasmid R1. Kis prevents the inhibition of E. coli cell growth caused by the RNA cleavage activity of Kid. Overproduction of MazE, the chromosome-encoded homologue of Kis, has been demonstrated to neutralize Kid toxicity to a certain extent in the absence of native Kis. Here, we show that a high structural similarity exists between these antitoxins, using NMR spectroscopy. We report about the interactions between Kid and Kis that are responsible for neutralization of Kid toxicity and enhance autoregulation of parD transcription. Native macromolecular mass spectrometry data demonstrate that Kid and Kis form multiple complexes. At Kis:Kid ratios equal to or exceeding 1:1, as found in vivo in a plasmid-containing cell, various complexes are present, ranging from Kid(2)-Kis(2) tetramer up to Kis(2)-Kid(2)-Kis(2)-Kid(2)-Kis(2) decamer. When Kid is in excess of Kis, corresponding to an in vivo situation immediately after loss of the plasmid, the Kid(2)-Kis(2)-Kid(2) heterohexamer is the most abundant species. NMR chemical shift and intensity perturbations in the (1)H (15)N HSQC spectra of Kid and Kis, observed when titrating the partner protein, show that the interaction sites of Kid and Kis resemble those within the previously reported MazF(2)-MazE(2)-MazF(2) complex. Furthermore, we demonstrate that Kid(2)-MazE(2) tetramers can be formed via weak interactions involving a limited part of the Kis-binding residues of Kid. The functional roles of the identified Kid-Kis and Kid-MazE interaction sites and complexes in toxin neutralization and repression of transcription are discussed.
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http://dx.doi.org/10.1002/prot.21254DOI Listing
April 2007

Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry.

Anal Chem 2006 Nov;78(21):7473-83

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.
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http://dx.doi.org/10.1021/ac061039aDOI Listing
November 2006

Resolving stoichiometries and oligomeric states of glutamate synthase protein complexes with curve fitting and simulation of electrospray mass spectra.

Rapid Commun Mass Spectrom 2006 ;20(16):2490-6

Utrecht University, Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

A complicating factor in analyzing electrospray ionization mass spectra of intact macromolecular heterogeneous protein complexes is the potential overlap of ions from different species present in solution. Therefore, it is often not possible to assign all ion signals. With the aim of allowing the more efficient and comprehensive analysis of very complex mass spectra of intact heterogeneous protein complexes we developed a software program: SOMMS. The program uses simple user input parameters together with Gaussian curve fitting to simulate putative mass spectra of protein (sub)complexes within a specified charge state window. In addition, the program can simulate spectra for heterogeneous protein complexes using bi- and multinomial distributions and it can calculate zero-charge spectra and relatively quantify the abundance of each component in a mixture. As a proof of concept we analyzed the complex mass spectra of alpha-glutamate synthase and alphabeta-glutamate synthase from Azosprillum brasilense. Using our program we could determine that alpha-glutamate synthase is in equilibrium between its dimeric, tetrameric, hexameric and dodecameric conformation, whereas alphabeta-glutamate synthase forms up to 15 different heterooligomeric assemblies composed of alpha- and beta-subunits. Thus, SOMMS allows resolving stoichiometries and oligomeric states of protein complexes even from very complicated mass spectra. These complexes could not be assigned by using maximum entropy calculations. We compared our mass spectrometry data on glutamate synthases with available X-ray, small-angle X-ray scattering and size-exclusion chromatography data.
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http://dx.doi.org/10.1002/rcm.2620DOI Listing
November 2007

Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.

J Biol Chem 2006 Sep 17;281(37):27378-88. Epub 2006 Jul 17.

Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, the Netherlands.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.
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http://dx.doi.org/10.1074/jbc.M605549200DOI Listing
September 2006

Probing genuine strong interactions and post-translational modifications in the heterogeneous yeast exosome protein complex.

Mol Cell Proteomics 2006 Sep 7;5(9):1581-92. Epub 2006 Jul 7.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

The characterization of heterogeneous multicomponent protein complexes, which goes beyond identification of protein subunits, is a challenging task. Here we describe and apply a comprehensive method that combines a mild affinity purification procedure with a multiplexed mass spectrometry approach for the in-depth characterization of the exosome complex from Saccharomyces cerevisiae expressed at physiologically relevant levels. The exosome is an ensemble of primarily 3' --> 5' exoribonucleases and plays a major role in RNA metabolism. The complex has been reported to consist of 11 proteins in molecular mass ranging from 20 to 120 kDa. By using native macromolecular mass spectrometry we measured accurate masses (around 400 kDa) of several (sub)exosome complexes. Combination of these data with proteolytic peptide LC tandem mass spectrometry using a linear ion trap coupled to a FT-ICR mass spectrometer and intact protein LC mass spectrometry provided us with the identity of the different exosome components and (sub)complexes, including the subunit stoichiometry. We hypothesize that the observed complexes provide information about strongly and weakly interacting exosome-associated proteins. In our analysis we also identified for the first time phosphorylation sites in seven different exosome subunits. The phosphorylation site in the Rrp4 subunit is fully conserved in the human homologue of Rrp4, which is the only previously reported phosphorylation site in any of the human exosome proteins. The described multiplexed mass spectrometry-based procedure is generic and thus applicable to many different types of cellular molecular machineries even if they are expressed at endogenous levels.
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http://dx.doi.org/10.1074/mcp.M600043-MCP200DOI Listing
September 2006

CprK crystal structures reveal mechanism for transcriptional control of halorespiration.

J Biol Chem 2006 Sep 27;281(38):28318-25. Epub 2006 Jun 27.

Manchester Interdisciplinary Biocentre, P. O. Box 88, Manchester, M60 1QD, United Kingdom.

Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the ligand ortho-chlorophenolacetic acid and of reduced CprK in absence of this ligand. These structures reveal that highly specific binding of chlorinated, rather than the corresponding non-chlorinated, phenolic compounds in the NH(2)-terminal beta-barrels causes reorientation of these domains with respect to the central alpha-helix at the dimer interface. Unexpectedly, the COOH-terminal DNA-binding domains dimerize in the non-DNA binding state. We postulate the ligand-induced conformational change allows formation of interdomain contacts that disrupt the DNA domain dimer interface and leads to repositioning of the helix-turn-helix motifs. These structures provide a structural framework for further studies on transcriptional control by CRP-FNR homologs in general and of halorespiration regulation by CprK in particular.
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http://dx.doi.org/10.1074/jbc.M602654200DOI Listing
September 2006

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin--immunity protein complex.

J Am Soc Mass Spectrom 2006 Jul 19;17(7):983-994. Epub 2006 May 19.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA, Utrecht, The Netherlands.

To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure.
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http://dx.doi.org/10.1016/j.jasms.2006.03.005DOI Listing
July 2006

Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring.

J Am Chem Soc 2006 Apr;128(14):4694-702

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.
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http://dx.doi.org/10.1021/ja056756lDOI Listing
April 2006

Model for RNA binding and the catalytic site of the RNase Kid of the bacterial parD toxin-antitoxin system.

J Mol Biol 2006 Mar 27;357(1):115-26. Epub 2005 Dec 27.

Bijvoet Center for Biomolecular Research, Department of NMR Spectroscopy, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

The toxin Kid and antitoxin Kis are encoded by the parD operon of Escherichia coli plasmid R1. Kid and its chromosomal homologues MazF and ChpBK have been shown to inhibit protein synthesis in cell extracts and to act as ribosome-independent endoribonucleases in vitro. Kid cleaves RNA preferentially at the 5' side of the A residue in the nucleotide sequence 5'-UA(A/C)-3' of single-stranded regions. Here, we show that RNA cleavage by Kid yields two fragments with a 2':3'-cyclic phosphate group and a free 5'-OH group, respectively. The cleavage mechanism is similar to that of RNases A and T1, involving the uracil 2'-OH group. Via NMR titration studies with an uncleavable RNA mimic, we demonstrate that residues of both monomers of the Kid dimer together form a concatenated RNA-binding surface. Docking calculations based on the NMR chemical shifts, the cleavage mechanism and previously reported mutagenesis data provide a detailed picture of the position of the AUACA fragment within the binding pocket. We propose that residues D75, R73 and H17 form the active site of the Kid toxin, where D75 and R73 are the catalytic base and acid, respectively. The RNA sequence specificity is defined by residues T46, S47, A55, F57, T69, V71 and R73. Our data show the importance of these residues for Kid function, and the implications of our results for related toxins, such as MazF, CcdB and RelE, are discussed.
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http://dx.doi.org/10.1016/j.jmb.2005.12.033DOI Listing
March 2006

Structure-function studies on the complex iron-sulfur flavoprotein glutamate synthase: the key enzyme of ammonia assimilation.

Photosynth Res 2005 ;83(2):219-38

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Via Celoria 26, Milan 20131, Italy.

Glutamate synthases are complex iron-sulfur flavoproteins that participate in the essential ammonia assimilation pathway in microorganisms and plants. The recent determination of the 3-dimensional structures of the alpha subunit of the NADPH-dependent glutamate synthase form and of the ferredoxin-dependent enzyme of Synechocystis sp. PCC 6803 provides a framework for the interpretation of the functional properties of these enzymes, and highlights protein segments most likely involved in control and coordination of the partial catalytic activities of glutamate synthases, which take place at sites distant from each other in space. In this review, we focus on the current knowledge on structure-function relationships in glutamate synthases, and we discuss open questions on the mechanisms of control of the enzyme reaction and of electron transfer among the enzyme flavin cofactors and iron-sulfur clusters.
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http://dx.doi.org/10.1007/s11120-004-2438-zDOI Listing
September 2005

Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase.

J Biol Chem 2005 Sep 27;280(37):32115-21. Epub 2005 Jul 27.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University.

The NADPH-dependent dimeric flavoenzyme 4-hydroxyacetophenone monooxygenase (HAPMO) catalyzes Baeyer-Villiger oxidations of a wide range of ketones, thereby generating esters or lactones. In the current work, we probed HAPMO-coenzyme complexes present during the enzyme catalytic cycle with the aim to gain mechanistic insight. Moreover, we investigated the structural role of the nicotinamide coenzyme. For these studies, we used (i) wild type HAPMO, (ii) the R339A variant, which is active but has a low affinity toward NADPH, and (iii) the R440A variant, which is inactive but has a high affinity toward NADPH. Electrospray ionization mass spectrometry was used as the primary tool to directly observe noncovalent protein-coenzyme complexes in real time. These analyzes showed for the first time that the nicotinamide coenzyme remains bound to HAPMO during the entire catalytic cycle of the NADPH oxidase reaction. This may also have implications for other homologous Baeyer-Villiger monooxygenases. Together with the observations that NADP(+) only weakly interacts with oxidized enzyme and that HAPMO is mainly in the reduced form during catalysis, we concluded that NADP(+) interacts tightly with the reduced form of HAPMO. We also demonstrated that the association with the coenzyme is crucial for enzyme stability. The interaction with the coenzyme analog 3-aminopyridine adenine dinucleotide phosphate (AADP(+)) strongly enhanced the thermal stability of wild type HAPMO. This coenzyme-induced stabilization may also be important for related enzymes.
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http://dx.doi.org/10.1074/jbc.M503758200DOI Listing
September 2005

Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry.

Nucleic Acids Res 2005 Jun 13;33(10):e96. Epub 2005 Jun 13.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

A fast and direct method for the monitoring of enzymatic DNA hydrolysis was developed using electrospray ionization mass spectrometry. We incorporated the use of a robotic chip-based electrospray ionization source for increased reproducibility and throughput. The mass spectrometry method allows the detection of DNA fragments and intact non-covalent protein-DNA complexes in a single experiment. We used the method to monitor in real-time single-stranded (ss) DNA hydrolysis by colicin E9 DNase and to characterize transient non-covalent E9 DNase-DNA complexes present during the hydrolysis reaction. The mass spectra showed that E9 DNase interacts with ssDNA in the absence of a divalent metal ion, but is strictly dependent on Ni2+ or Co2+ for ssDNA hydrolysis. We demonstrated that the sequence selectivity of E9 DNase is dependent on the ratio protein:ssDNA or the ssDNA concentration and that only 3'-hydroxy and 5'-phosphate termini are produced. It was also shown that the homologous E7 DNase is reactive with Zn2+ as transition metal ion and that this DNase displays a different sequence selectivity. The method described is of general use to analyze the reactivity and specificity of nucleases.
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http://dx.doi.org/10.1093/nar/gni099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1150282PMC
June 2005

Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry.

Nat Methods 2005 May 21;2(5):371-6. Epub 2005 Apr 21.

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.
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http://dx.doi.org/10.1038/nmeth753DOI Listing
May 2005

Decharging of globular proteins and protein complexes in electrospray.

Chemistry 2005 Jan;11(3):960-8

Department of Biomolecular Mass Spectrometry, Utrecht Institute for Pharmaceutical Sciences and Bijvoet Center for Biomolecular Research, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.
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http://dx.doi.org/10.1002/chem.200400395DOI Listing
January 2005

Native protein mass spectrometry: from intact oligomers to functional machineries.

Curr Opin Chem Biol 2004 Oct;8(5):519-26

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.

The development of electrospray ionization coupled to mass spectrometry has enabled the analysis of very large intact protein complexes, even when they are held together by weak non-covalent interactions. Together with equally spectacular advances in mass spectrometric instrumentation, a new field has emerged, termed native protein mass spectrometry, which focuses on the structural and functional analysis of the dynamics and interactions occurring in protein complexes. In the past two years, several important progressive steps in technologies have been reported that have led to exciting applications ranging from the detailed study of equilibria between different quaternary structures as influenced by environmental changes or binding of substrates or cofactors, to the analysis of intact nano-machineries, such as whole virus particles, proteasomes and ribosomes.
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http://dx.doi.org/10.1016/j.cbpa.2004.08.006DOI Listing
October 2004

Crystal structure of E.coli alcohol dehydrogenase YqhD: evidence of a covalently modified NADP coenzyme.

J Mol Biol 2004 Sep;342(2):489-502

Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS and Universités d'Aix-Marseille I and II, 31 chemin J. Aiguier, F-13402 Marseille Cedex 20, France.

In the course of a structural genomics program aiming at solving the structures of Escherichia coli open reading frame (ORF) products of unknown function, we have determined the structure of YqhD at 2.0A resolution using the single wavelength anomalous diffraction method at the Pt edge. The crystal structure of YqhD reveals that it is an NADP-dependent dehydrogenase, a result confirmed by activity measurements with several alcohols. The current interpretation of our findings is that YqhD is an alcohol dehydrogenase (ADH) with preference for alcohols longer than C(3). YqhD is a dimer of 2x387 residues, each monomer being composed of two domains, a Rossmann-type fold and an alpha-helical domain. The crystals contain two dimers in the asymmetric unit. While one of the dimers contains a cofactor in both subunits, only one of the subunits in the second dimer contains it, making it possible to compare bound and unbound active sites. The active site contains a Zn atom, as verified by EXAFS on the crystals. The electron density maps of NADP revealed modifications of the nicotinamide ring by oxygen atoms at positions 5 and 6. Further analysis by electrospray mass spectrometry and comparison with the mass spectra of NADP and NADPH revealed the nature of the modification and the incorporation of two hydroxyl moieties at the 5 and 6 position in the nicotinamide ring, yielding NADPH(OH)(2). These modifications might be due to oxygen stress on an enzyme, which would functionally work under anaerobic conditions.
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http://dx.doi.org/10.1016/j.jmb.2004.07.034DOI Listing
September 2004

Investigation of intact protein complexes by mass spectrometry.

Mass Spectrom Rev 2004 Sep-Oct;23(5):368-89

Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, The Netherlands.

Mass spectrometry has grown in recent years to a well-accepted and increasingly important complementary technique in structural biology. Especially electrospray ionization mass spectrometry is well suited for the detection of non-covalent protein complexes and their interactions with DNA, RNA, ligands, and cofactors. Over the last decade, significant advances have been made in the ionization and mass analysis techniques, which makes the investigation of even larger and more heterogeneous intact assemblies feasible. These technological developments have paved the way to study intact non-covalent protein-protein interactions, assembly and disassembly in real time, subunit exchange, cooperativity effects, and effects of cofactors, allowing us a better understanding of proteins in cellular processes. In this review, we describe some of the latest developments and several highlights.
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http://dx.doi.org/10.1002/mas.10081DOI Listing
September 2004