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

PloS One
Mykhaylo O DebelyyAndreas Conzelmann

Abstract

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).

References

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Citations

Mar 5, 2019·Annual Review of Biochemistry·Merav BraitbardNir Kalisman
Oct 30, 2020·Analytical Chemistry·James E KeenerMichael T Marty

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Datasets Mentioned

BETA
PXD006707

Methods Mentioned

BETA
X-ray
acetylation

Software Mentioned

Proteome Software
HHPRED
Scaffold
StavroX
Proteome
pLink
TOPCONS
Mascot
Proteome Software Scaffold
xVis

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