Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Happersberger, H. Peter; Stapleton, Janet; Cowgill, Cynthia; Glocker, Michael O.

doi:10.1002/(sici)1097-0134(1998)33:2+<50::aid-prot7>3.0.co;2-r

Cited by 13 publications

(3 citation statements)

References 41 publications

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“…Furthermore they have been used to identify bis‐sulfhydryl groups in reduced proteins which are suitably spaced to form disulfide bridges. The recently introduced arsonous acid derivative MEL has some advantages for protein modification, especially in combination with MS analysis, because of its higher water solubility and larger mass increment upon modification [25,41]. CaN was reacted with different amounts of PAO and MEL at pH 8.05.…”

Section: Resultsmentioning

confidence: 99%

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

Bogumil¹,

Namgaladze²,

Schaarschmidt³

et al. 2000

European Journal of Biochemistry

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Calcineurin (CaN) is a Ca 2+ -and calmodulin (CaM)-dependent serine/threonine phosphatase containing a dinuclear Fe±Zn center in the active site. Recent studies have indicated that CaN is a possible candidate for redox regulation. The inactivation of bovine brain CaN and of the catalytic CaN A-subunit from Dictyostelium by the vicinal dithiol reagents phenylarsine oxide (PAO) and melarsen oxide (MEL) and by H 2 O 2 was investigated. PAO and MEL inhibited CaN with an IC 50 of 3±8 mm and the inactivation was reversed by 2,3-dimercapto-1-propane sulfonic acid. The treatment of isolated CaN with hydrogen peroxide resulted in a concentration-dependent inactivation. Analysis of the free thiol content performed on the H 2 O 2 inactivated enzyme demonstrated that only two or three of the 14 Cys residues in CaN are modified. The inactivation of CaN by H 2 O 2 could be reversed with 1,4-dithiothreitol and with the dithiol oxidoreductase thioredoxin. We propose that a bridging of two closely spaced Cys residues in the catalytic CaN A-subunit by PAO/MEL or the oxidative formation of a disulfide bridge by H 2 O 2 involving the same Cys residues causes the inactivation. Our data implicate a possible involvement of thioredoxin in the redox control of CaN activity under physiological conditions. The low temperature EPR spectrum of the native enzyme was consistent with a Fe 3 ±Zn 2 dinuclear centre. Upon H 2 O 2 -mediated inactivation of the enzyme no significant changes in the EPR spectrum were observed ruling out that Fe 2+ is present in the active enzyme and that the dinuclear metal centre is the target for the oxidative inactivation of CaN.

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Section: Resultsmentioning

confidence: 99%

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

Bogumil¹,

Namgaladze²,

Schaarschmidt³

et al. 2000

European Journal of Biochemistry

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“…Given the high reactivity of the Cys thiol group, specific labeling reagents were also developed to follow protein folding in addition to footprinting a static state . An early application addressed slow events in the folding of recombinant human macrophage-colony stimulating-factor β, which takes tens of hours to complete . Melarsen oxide ( p -(4,6-diamino-1,3,5-triazin-2-yl)aminophenylarsonous acid) used in this study footprints Cys in 10 min, which is sufficiently fast for this slow folding system but far too slow to footprint most protein folding.…”

Section: Applications Of Targeted-labeling Reagentsmentioning

confidence: 99%

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

2020

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Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

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“…This approach is especially useful for comparing two different states of the same protein, and this comparison can reveal changes in the protection of specific residues that result from changes in conformation and/or complex formation. Noteworthy applications of the approach, as in case with limited proteolysis, are the characterization of folding intermediates − and the determination of protein interaction interfaces and of antibody epitopes for protein antigens, in particular. ,, We used this approach to study the conformational changes involved in the misfolding of the prion, α-synuclein, and tau proteins and for the characterization of the conformational changes that occur upon ligand-induced disorder-to-order protein structure transitions. ,− In a differential modification experiment, surface-modification experiments are performed in parallel for two states of the protein, and differences in the reactivities of specific amino acid residues, reflecting differences in the degree of protection or exposure, can be detected by mass spectrometry. While label-free quantitation can give an estimate of the relative amounts of modification under the two conditions, isotopically coded modification reagentswhich behave identically during the modification reaction and during mass spectrometric analysiscan provide a more accurate estimate of any changes in protection.…”

Section: Covalent Modificationmentioning

confidence: 99%

Protein Chemistry Combined with Mass Spectrometry for Protein Structure Determination

Petrotchenko

Borchers

2021

Chem. Rev.

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The advent of soft-ionization mass spectrometry for biomolecules has opened up new possibilities for the structural analysis of proteins. Combining protein chemistry methods with modern mass spectrometry has led to the emergence of the distinct field of structural proteomics. Multiple protein chemistry approaches, such as surface modification, limited proteolysis, hydrogen–deuterium exchange, and cross-linking, provide diverse and often orthogonal structural information on the protein systems studied. Combining experimental data from these various structural proteomics techniques provides a more comprehensive examination of the protein structure and increases confidence in the ultimate findings. Here, we review various types of experimental data from structural proteomics approaches with an emphasis on the use of multiple complementary mass spectrometric approaches to provide experimental constraints for the solving of protein structures.

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Characterization of the folding pathway of recombinant human macrophage-colony stimulating-factor β (rhM-CSF β) by bis-cysteinyl modification and mass spectrometry

Cited by 13 publications

References 41 publications

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

Inactivation of calcineurin by hydrogen peroxide and phenylarsine oxide

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

Protein Chemistry Combined with Mass Spectrometry for Protein Structure Determination

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