Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.

Détails

Ressource 1Télécharger: journal.pone.0186840.pdf (3787.76 [Ko])
Etat: Serval
Version: Final published version
ID Serval
serval:BIB_76684B3AA058
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Chemical crosslinking and mass spectrometry to elucidate the topology of integral membrane proteins.
Périodique
PLoS One
Auteur(s)
Debelyy M.O., Waridel P., Quadroni M., Schneiter R., Conzelmann A.
ISSN
1932-6203 (Electronic)
ISSN-L
1932-6203
Statut éditorial
Publié
Date de publication
2017
Peer-reviewed
Oui
Volume
12
Numéro
10
Pages
e0186840
Langue
anglais
Résumé
Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).

Mots-clé
Cross-Linking Reagents/chemistry, Mass Spectrometry, Membrane Proteins/chemistry, Membrane Proteins/metabolism, Saccharomyces cerevisiae/chemistry, Saccharomyces cerevisiae/metabolism, Saccharomyces cerevisiae Proteins/chemistry, Saccharomyces cerevisiae Proteins/metabolism
Pubmed
Web of science
Open Access
Oui
Création de la notice
16/11/2017 8:52
Dernière modification de la notice
08/05/2019 20:33
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