Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Pan, Yan; Brown, Leonid S.; Konermann, Lars

doi:10.1016/j.jasms.2010.08.004

Cited by 19 publications

(22 citation statements)

References 82 publications

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“…Such an approach may be preferable to the use of oxidative labeling data for the post-processing assessment of competing models. The mutational introduction of additional easily oxidizable residues in strategic locations will further enhance the resolution of the approach used here [61]. In addition, it may be possible to conduct labeling experiments on IMPs in their natural bilayer environment (possibly even in vivo), instead of relying on purified proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Pulsed ⋅OH labeling was performed using a two-syringe continuous-flow device similar to that described previously [61]. Syringe 1 contained 20 μM WaaL in 0.05 % DM and 150 mM NaCl, 2.5 mM imidazole, 50 mM phosphate buffer (pH 8), 2% glycerol, and 30 mM glutamine, while syringe 2 contained 0.3% (vol/vol) H 2 O 2 and 0.05% DM in phosphate buffer (pH 8).…”

Section: Oxidative Labelingmentioning

confidence: 99%

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Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Pan

Ruan

Valvano

et al. 2012

J. Am. Soc. Mass Spectrom.

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Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ⋅OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424. Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ⋅OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

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Section: Discussionmentioning

confidence: 99%

Section: Oxidative Labelingmentioning

confidence: 99%

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Pan

Ruan

Valvano

et al. 2012

J. Am. Soc. Mass Spectrom.

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“…However, there is also precedent for Met oxidation causing large-scale changes in protein structure (Griffiths and Cooney 2002;Wolschner et al 2009;Pan et al 2010;Marondedze et al 2013). If this were the case, it might easily lead to exposure of a previously cryptic phosphorylation site or to hide a previously exposed site.…”

Section: Crosstalk Between Met Oxidation and O-phosphorylationmentioning

confidence: 99%

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

Rao

Thelen

Miernyk

2015

Cell Stress and Chaperones

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Oxidation of methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible posttranslational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/ cells from oxidative stress. However, Met oxidation has been established as a key mechanism for direct regulation of a wide range of protein functions and cellular processes. Furthermore, recent reports suggest an interaction between Met oxidation and O-phosphorylation. Such interactions are a potentially direct interface between redox sensing and signaling, and cellular protein kinase/phosphatase-based signaling. Herein, we describe the current state of Met oxidation research, provide some mechanistic insight into crosstalk between these two major posttranslational modifications, and consider the evolutionary significance and regulatory potential of this crosstalk.

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“…This oxidative labeling strategy has been applied for monitoring conformational changes and ligand binding of several membrane proteins under equilibrium conditions. [43][44][45][46][47] Met residues are particularly useful labeling sites 45,46,48 due to their high propensity to form methionine sulfoxide (MetO). 39 Hydroxyl radical may be formed in different ways, 39 for example, by using an excimer laser for the photolysis of H 2 O 2 .…”

Section: Introductionmentioning

confidence: 99%

“…67 This is consistent with H/D exchange data, which suggest partial helix unraveling, particularly on the cytoplasmic side. 48,68 Covalent labeling indicates that helices A and D (Fig. 1, highlighted in orange) are most disordered and likely extruded from the residual protein core.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

Pan

Brown

Konermann

2011

Journal of Molecular Biology

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Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Cited by 19 publications

References 82 publications

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Validation of Membrane Protein Topology Models by Oxidative Labeling and Mass Spectrometry

Convergent signaling pathways—interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

Kinetic Folding Mechanism of an Integral Membrane Protein Examined by Pulsed Oxidative Labeling and Mass Spectrometry

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