NDLI: Topology of OxlT, the Oxalate Transporter of Oxalobacter formigenes, Determined by Site-Directed Fluorescence Labeling

Content Provider	PubMed Central
Author	Ye, Liwen Jia, Zhenzhen Jung, Thomas Maloney, Peter C.
Copyright Year	2001
Abstract	The topology of OxlT, the oxalate:formate exchange protein of Oxalobacter formigenes, was established by site-directed fluorescence labeling, a simple strategy that generates topological information in the context of the intact protein. Accessibility of cysteine to the fluorescent thiol-directed probe Oregon green maleimide (OGM) was examined for a panel of 34 single-cysteine variants, each generated in a His9-tagged cysteine-less host. The reaction with OGM was readily scored by examining the fluorescence profile after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of material purified by Ni2+-linked affinity chromatography. A position was assigned an external location if its single-cysteine derivative reacted with OGM added to intact cells; a position was designated internal if OGM labeling required cell lysis. We also showed that labeling of external, but not internal, positions was blocked by prior exposure of cells to the impermeable and nonfluorescent thiol-specific agent ethyltrimethylammonium methanethiosulfonate. Of the 34 positions examined in this way, 29 were assigned unambiguously to either an internal or external location; 5 positions could not be assigned, since the target cysteine failed to react with OGM. There was no evidence of false-positive assignment. Our findings document a simple and rapid method for establishing the topology of a membrane protein and show that OxlT has 12 transmembrane segments, confirming inferences from hydropathy analysis.
Related Links	http://dx.doi.org/10.1128/jb.183.8.2490-2496.2001
Ending Page	2496
Page Count	7
Starting Page	2490
File Format	PDF
ISSN	00219193
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	8
Volume Number	183
Language	English
Publisher	American Society for Microbiology
Publisher Date	2001-04-15
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Molecular Biology Microbiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling (2001)

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