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 (G₁₄₀LASG₁₄₄ and S₃₃₆DIFG₃₄₀) 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