Defining the Interacting Regions between Apomyoglobin and Lipid Membrane by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Man, Petr; Montagner, Caroline; Vernier, Grégory; Dublet, Bernard; Chenal, Alexandre; Forest, Éric; Forge, Vincent

doi:10.1016/j.jmb.2007.02.014

Cited by 42 publications

(41 citation statements)

References 40 publications

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“…According to the currently accepted "helical anchor" model, an amphipathic ␣-helix packs its aliphatic side against the hydrophobic protein core in the soluble state and rotates it to present this side to the membrane in the bound state while the charge side of the helix faces the lipid headgroup region (22)(23)(24)(25)(26). We have previously postulated that the amphipathic ␣2-helix is an important element to determine the binding of PimA to the membrane (11,14).…”

Section: Labeled Suv Typementioning

confidence: 99%

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

Rodrigo-Unzueta

Martínez

Comino

et al. 2016

Journal of Biological Chemistry

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Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannoside, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of membrane-associated glycosyltransferases for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here, we determined that PimA preferentially binds to negatively charged phosphatidylmyo-inositol substrate and non-substrate membrane model systems (small unilamellar vesicle) through its N-terminal domain, inducing an important structural reorganization of anionic phospholipids. By using a combination of single-point mutagenesis, circular dichroism, and a variety of fluorescence spectroscopy techniques, we determined that this interaction is mainly mediated by an amphipathic ␣-helix (␣2), which undergoes a substantial conformational change and localizes in the vicinity of the negatively charged lipid headgroups and the very first carbon atoms of the acyl chains, at the PimA-phospholipid interface. Interestingly, a flexible region within the N-terminal domain, which undergoes ␤-strand-to-␣-helix and ␣-helix-to-␤-strand transitions during catalysis, interacts with anionic phospholipids; however, the effect is markedly less pronounced to that observed for the amphipathic ␣2, likely reflecting structural plasticity/variability. Altogether, we propose a model in which conformational transitions observed in PimA might reflect a molten globule state that confers to PimA, a higher affinity toward the dynamic and highly fluctuating lipid bilayer.

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Section: Labeled Suv Typementioning

confidence: 99%

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

Rodrigo-Unzueta

Martínez

Comino

et al. 2016

Journal of Biological Chemistry

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“…Protein hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) is a highly sensitive technique to monitor protein backbone dynamics, solvent accessibility, and protein-protein or protein-ligand interactions [1][2][3][4][5][6]. In contrast to high-resolution structural techniques it has much smaller requirements in terms of sample amount and concentration.…”

Section: Hydrogen/deuterium Exchange Of Proteinsmentioning

confidence: 99%

“…Therefore HXMS represents a complementary technique to NMR or protein X-ray crystallography. Its speed and possibility of automation make it a technique of choice in cases where the classical structural techniques meet their limitations [5,10]. The main weakness of HXMS, its relatively low spatial resolution, can be addressed by the use of different proteases, dissociation techniques or site directed mutagenesis [11][12][13][14][15][16][17][18][19][20].…”

Section: Hydrogen/deuterium Exchange Of Proteinsmentioning

confidence: 99%

“…It becomes especially useful when several conditions (e.g., different pH values, various salt concentrations) have to be followed [10]. It also helps in cases where time correction has to be done, e.g., H/D performed at different pH values [5,31]. The result is represented by a simple table showing the times for mixing of the reaction and collection of aliquots.…”

Section: H/d Experiments Plannermentioning

confidence: 99%

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MSTools—Web based application for visualization and presentation of HXMS data

Kavan

Man

2011

International Journal of Mass Spectrometry

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136

107

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“…The combined use of alternative acidic proteases with different specificities is another way to improve the method's resolution. Our laboratory has developed the use of commercial-type XIII and XVIII fungal proteases [7,8] that are now routinely used to improve the resolution of DXMS studies [9,10]. However, the presence of impurities requires high enzyme/protein ratios (10.5:1 (wt/wt) for protease type XIII and 17:1 for protease type XVIII) [7].…”

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confidence: 99%

Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

Marcoux

Thierry

Vivès

et al. 2010

J. Am. Soc. Mass Spectrom.

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Structural studies of proteins by hydrogen/deuterium exchange coupled to mass spectrometry (DXMS) require the use of proteases working at acidic pH and low temperatures. The spatial resolution of this technique can be improved by combining several acidic proteases, each generating a set of different peptides. Three commercial aspartic proteases are used, namely, pepsin, and proteases XIII and XVIII. However, given their low purity, high enzyme/protein ratios have to be used with proteases XIII and XVIII. In the present work, we investigate the activity of two alternative acidic proteases from Plasmodium falciparum under different pH and temperature conditions. Peptide mapping of four different proteins after digestion with pepsin, plasmepsin 2 (PSM2), and plasmepsin 4 (PSM4) were compared. PSM4 is inactive at pH 2.2 and 0°C, making it unusable for DXMS studies. However, PSM2 showed low but reproducible activity under DXMS conditions. It displayed no substrate specificity and, like pepsin, no strict sequence specificity. Altogether, these results show that PSM2 but not PSM4 is a potential new tool for DXMS studies. (J Am Soc Mass Spectrom 2010, 21, 76 -79)

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Defining the Interacting Regions between Apomyoglobin and Lipid Membrane by Hydrogen/Deuterium Exchange Coupled to Mass Spectrometry

Cited by 42 publications

References 40 publications

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

Molecular Basis of Membrane Association by the Phosphatidylinositol Mannosyltransferase PimA Enzyme from Mycobacteria

MSTools—Web based application for visualization and presentation of HXMS data

Investigating alternative acidic proteases for H/D exchange coupled to mass spectrometry: Plasmepsin 2 but not plasmepsin 4 is active under quenching conditions

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