TY - JOUR
T1 - Topology of OxlT, the oxalate transporter of Oxalobacter formigenes, determined by site-directed fluorescence labeling
AU - Ye, L.
AU - Jia, Z.
AU - Jung, T.
AU - Maloney, P. C.
PY - 2001
Y1 - 2001
N2 - 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.
AB - 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.
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U2 - 10.1128/JB.183.8.2490-2496.2001
DO - 10.1128/JB.183.8.2490-2496.2001
M3 - Article
C2 - 11274108
AN - SCOPUS:0035089130
SN - 0021-9193
VL - 183
SP - 2490
EP - 2496
JO - Journal of bacteriology
JF - Journal of bacteriology
IS - 8
ER -