2 research outputs found
Closure of the Cytoplasmic Gate Formed by TM5 and TM11 during Transport in the Oxalate/Formate Exchanger from <i>Oxalobacter formigenes</i>
OxlT, the oxalate/formate exchanger
of Oxalobacter
formigenes, is a member of the major facilitator superfamily
of transporters. In the present work, substrate (oxalate) was found
to enhance the reactivity of the cysteine mutant S336C on the cytoplasmic
end of helix 11 to methanethiosulfonate ethyl carboxylate. In addition,
S336C is found to spontaneously cross-link to S143C in TM5 in either
native or reconstituted membranes under conditions that support transport.
Continuous wave EPR measurements are consistent with this result and
indicate that positions 143 and 336 are in close proximity in the
presence of substrate. These two residues are localized within helix
interacting GxxxG-like motifs (G<sub>140</sub>LASG<sub>144</sub> and
S<sub>336</sub>DIFG<sub>340</sub>) at the cytoplasmic poles of TM5
and TM11. Pulse EPR measurements were used to determine distances
and distance distributions across the cytoplasmic or periplasmic ends
of OxlT and were compared with the predictions of an inside-open homology
model. The data indicate that a significant population of transporter
is in an outside-open configuration in the presence of substrate;
however, each end of the transporter exhibits significant conformational
heterogeneity, where both inside-open and outside-open configurations
are present. These data indicate that TM5 and TM11, which form part
of the transport pathway, transiently close during transport and that
there is a conformational equilibrium between inside-open and outside-open
states of OxlT in the presence of substrate
Nanochannel-Based Single Molecule Recycling
We present a method for measuring the fluorescence from
a single
molecule hundreds of times without surface immobilization. The approach
is based on the use of electroosmosis to repeatedly drive a single
target molecule in a fused silica nanochannel through a stationary
laser focus. Single molecule fluorescence detected during the transit
time through the laser focus is used to repeatedly reverse the electrical
potential controlling the flow direction. Our method does not rely
on continuous observation and therefore is less susceptible to fluorescence
blinking than existing fluorescence-based trapping schemes. The variation
in the turnaround times can be used to measure the diffusion coefficient
on a single molecule level. We demonstrate the ability to recycle
both proteins and DNA in nanochannels and show that the procedure
can be combined with single-pair Förster energy transfer. Nanochannel-based
single molecule recycling holds promise for studying conformational
dynamics on the same single molecule in solution and without surface
tethering