Detection of a Monomeric Intermediate Associated with Dimerization of Protein Hu by Mass Spectrometry

Vis, Hans; Heinemann, Udo; Dobson, Christopher M.; Robinson, Carol V.

doi:10.1021/ja9811187

Cited by 59 publications

(63 citation statements)

References 25 publications

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“…8 were generally duplicated within ∼5%). Further, it has been previously observed that the presence of nonnative or oligomeric forms of the protein can alter in the charge state distribution seen with ESI-MS relative to that of the native, monomeric protein (Katta and Chait, 1993;Vis et al, 1998). Although the instrument parameters here were not optimized for the purpose of observing noncovalent interactions, all of the spectra exhibited the same charge state distribution, providing no evidence for any oligomeric or otherwise nonnative forms of lysozyme at the time of MS analysis.…”

Section: Mass Spectrometrymentioning

confidence: 89%

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Tobler

Sherman

Fernandez

2000

Biotechnol. Bioeng.

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Section: Mass Spectrometrymentioning

confidence: 89%

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Tobler

Sherman

Fernandez

2000

Biotechnol. Bioeng.

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“…In solution, protein assembly is often dependent on the ionic strength and/or type of salt (46). In all the above reported data the ammonium acetate concentration was 50 mM.…”

Section: Cofactor-induced Oligomerization Of Apovao H61t-inmentioning

confidence: 96%

Cofactor-dependent Assembly of the Flavoenzyme Vanillyl-alcohol Oxidase

Tahallah¹,

Heuvel²,

Berg³

et al. 2002

Journal of Biological Chemistry

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The oligomerization of the flavoprotein vanillyl-alcohol oxidase (VAO) and its site-directed mutant H61T was studied by mass spectrometry. Native VAO has a covalently bound FAD and forms primarily octameric assemblies of 507 kDa. H61T is purified as a FAD-free apoprotein and mainly exists as a dimeric species of 126 kDa. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization, although association of H61T dimers seems not to be crucial for enzyme activity. Reconstitution of H61T with the cofactor analog 5-ADP also promotes octamerization. FMN on the other hand, interacts with apoH61T without stimulating dimer association. These results are in line with observations made for several other flavoenzymes, which contain a Rossmann fold. Members of the VAO flavoprotein family do not contain a Rossmann fold but do share two conserved loops that are responsible for binding the pyrophosphate moiety of FAD. Therefore, the observed FAD-induced oligomerization might be general for this family. We speculate that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO species are transmitted to the protein surface, promoting oligomerization.Riboflavin (vitamin B 2 ) derivatives such as FAD and FMN are essential components of all living organisms, serving as cofactors for numerous proteins with diverse functions, ranging from electron transport, redox catalysis, oxygen activation, and light emission to DNA repair (1). For most reported flavoproteins, the flavin cofactor is noncovalently bound, although ϳ10% of human cellular FAD is covalently linked to enzymes like monoamine oxidase and succinate dehydrogenase (2, 3). Many noncovalent flavoproteins can be reversibly dissociated into their constituents the apoprotein and flavin prosthetic group (4 -6). Reconstitution of the holoprotein with either artificial (1,7,8), enzymatically modified (9), or isotopically enriched flavin analogs (4, 6, 10) then allows us to gain insight into the role of the protein in redox catalysis. For several flavoenzymes, binding of the FAD cofactor induces subunit association and improves the resistance of the protein to thermal and chemical denaturation. Thus, for glucose oxidase (11), D-amino-acid oxidase (12), and lipoamide dehydrogenase (13-15), it was shown that rapid FAD binding to the monomeric apoprotein is followed by relatively slow dimerization and regain of catalytic activity. For bacterial butyryl-CoA dehydrogenase on the other hand, it was revealed that optimal reconstitution of tetrameric holoenzyme from dimeric apoenzyme and FAD requires the presence of CoA ligands (16).For obvious reasons, little is known about the role of flavin binding in the assembly of covalent flavoenzymes (17). Covalent flavinylation mostly occurs via a histidine residue, but enzymes with cysteinyl-flavin, tyrosyl-flavin, and threonyl-flavin have been described as well (3). Vanillyl-alcohol oxidase (VAO) 1 from Penicillium simplicissimum is a covalent flavoprotein, whereby the FAD is bound via ...

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“…Such observations of complexes at high salt concentrations with nanospray when nothing is observed under low salt conditions have been made for a number of complexes at Manitoba, and have been reported also for the HU protein. 53 Nevertheless, under the most favorable conditions the ions for the dimer and the [dimer C 1 lipid] were always accompanied by ions of similar or greater abundance for the monomeric protein.…”

Section: Monomeric and Polymeric States Of Csact And Cs-csact Complexesmentioning

confidence: 98%

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties

Faull¹,

Whitelegge²,

Higginson³

et al. 1999

J. Mass Spectrom.

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Detection of a Monomeric Intermediate Associated with Dimerization of Protein Hu by Mass Spectrometry

Cited by 59 publications

References 25 publications

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Cofactor-dependent Assembly of the Flavoenzyme Vanillyl-alcohol Oxidase

Cerebroside sulfate activator protein (Saposin B): chromatographic and electrospray mass spectrometric properties

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