Development of an spFRET method to measure structure changes in ion exchange proteins.
OBJECTIVE
Understanding the mechanism of tightly coupled ion exchange proteins, important effectors of cell volume regulation and other physiologically important transport processes requires means to observe dynamic changes in structure during the transport cycle. As a step towards this goal, we have applied single-pair fluorescence resonance energy transfer to a monomeric bacterial oxalate-formate exchanger (OxlT).


METHODS
A His-9 tagged OxlT mutant containing two cysteines at positions 17 and 224 was labelled with cyanine dye maleimides (Cy3 donor and Cy5 acceptor) and attached to glass coverslips for measurements of donor and acceptor emission from single molecules, as described (P. Pal et al. Biophys J89, L11, 2005).


RESULTS
Time-series data from 20 spots containing donor and acceptor provided evidence for single-pair energy transfer. From the efficiency of energy transfer, the mean donor-acceptor distance was determined to be 44.2 A. Considering the size of the probes, this is in good agreement with the Calpha distance of 39.6 A for the corresponding sites found in the OxlT structural (homology) model (Q. Yang et al. Proc Natl Acad Sci102, 8513, 2005).


CONCLUSIONS
These results demonstrate the feasibility of single-pair fluorescence resonance energy transfer to measure distances between known sites in single OxlT molecules. This technique provides a potential means to test models for transport-related conformational changes, as well as to detect real-time structure alterations during the catalytic transport cycle.
Lesoine
J F
JF
Institute of Optics, University of Rochester, Rochester, NY 14627, USA. lesoine@optics.rochester.edu
Holmberg
B
B
Maloney
P
P
Wang
X
X
Novotny
L
L
Knauf
P A
PA
D002232:Carbocyanines / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D048429:Cell Size; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007474:Ion Exchange; D017136:Ion Transport; D026901:Membrane Transport Proteins / Q000032:analysis*; D018613:Microscopy, Confocal; D008956:Models, Chemical
D002232:Carbocyanines; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; C089187:cyanine dye 3; C085321:cyanine dye 5