Conformational Dynamics of Lipid-Reconstituted LeuT Studied at the Single Vesicle and Single Molecule Level

Abstract

Conformational changes are an essentially prerequisite for the function of transmembrane(TM) proteins. Ensemble studies typically average dynamics of the conformational changes that consequently become obscure. Therefore an approach for revealing the dynamics of conformational changes is to study each protein at the single molecule (SM) level. At present only two reports have addressed the conformational dynamics of any TM protein at SM level1,2 reflecting the difficulties and importance of these experiments. Proteins in these experiments were solubilized in detergent micelles. Here we investigate conformational dynamics of the Leucine transporter(LeuT) reconstituted in lipid vesicles, to understand the influence of the membrane on the transporter. We are focusing on single vesicle and SM microscopy measurements of allosteric transitions and oscillations between different states of the sixth TM helix (TM6) of LeuT.We have developed a unique strategy3,4,5,6 for immobilization of TM proteins under conditions that minimize non-specific interactions with the surface and thus minimize denaturation. We reconstitute membrane proteins into vesicles, which are anchored on a Neutravidin coated surface with biotinilated lipids. In this manner vesicle thus serves as a 3D scaffold that minimizes protein-surface interactions. By employing this method we have successfully reconstituted LeuT. The protein is labeled with the tetramethylrhodamine (TMR) dye on TM6 (at position 192C), which is quenched by Histidine (position 7H) when LeuT is in an inactive conformation. Variation in distance or orientation between the quencher and TMR, which are induced by the conformational changes of the protein during binding of substrate affect fluorescence signal intensity of the TMR. The dynamics of conformational changes are monitored by Total Internal Reflection microscopy at the single vesicle and the SM level.