EmrE is anEscherichia coli H ؉ -coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE.

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein.The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 µM with the protein concentration of 5 µM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.

The recently suggested antiparallel topology of EmrE has intriguing implications for many aspects of the biology of ioncoupled transporters. However, it is at odds with biochemical data that demonstrated the same topology for all protomers in the intact cell and with extensive cross-linking studies. To examine this apparent contradiction we chemically cross-linked dimers with a rigid bifunctional maleimide using Cys replacements at positions not permissible by an antiparallel topology. A purified cross-linked dimer binds substrate and transports it in proteoliposomes with kinetic constants similar to those of the non-cross-linked dimer. The cross-linked dimers do not interact with non-cross-linked dimers as judged from the fact that inactive mutants do not affect their activity (negative dominance). The results support the contention that EmrE with parallel topology is fully functional. We show that the detergents used in crystallization increase the fraction of monomers in solution. We suggest that the antiparallel orientation observed is a result of the arrangement of the monomers in the crystal. Functionality of EmrE with the suggested antiparallel orientation of the monomers remains to be characterized.Recent publications describing high resolution structures of transporters are changing a field that has been waiting avidly for such advancements (1-4). This welcome revolution provides fundamental insights for designing new biochemical/biophysical approaches and will deepen understanding of transport mechanisms.However, in some cases, as for the channel-forming peptide Gramicidin, for the ABC transporter MsbA, and for EmrE, an Escherichia coli ion-coupled multidrug transporter, different structures have been reported for the same protein, and it is not evident that these purportedly different conformations are physiologically relevant (5-8). The question is raised whether these are proteins with multiple conformations that fulfill functions yet unknown to us or whether the reported conformations are an experimental artifact created by the different milieu the proteins face when removed from their native environments. This may turn out to be especially critical for membrane proteins where we can only feebly mimic the original conditions after solubilization with detergents. A leading criterion at this stage should be whether the protein has some measurable function in the detergent-solubilized state.Two x-ray structures of EmrE have been published, and they are very different from each other (7,8). The presence of substrate in the second one may be responsible for the large differences between the two structures, although similar substrate-induced conformational changes were not observed in two-dimensional crystals (9, 10). The x-ray structure shows an asymmetric dimer with the protomers in an antiparallel topological orientation. This finding has obvious and exciting similarities to the internal structural repeat found in several membrane proteins such as aquaporins, ClC channel, and the neurotransmitter tran...

Changing practices in schools is a very complex endeavor. This paper is about new practices we prompted to foster collaboration and critical reasoning in science classrooms: the presentation of pictures representing different perspectives, small group synchronous argumentation, and moderation of synchronous argumentation. A CSCL tool helped in supporting synchronous argumentation through graphical representations of argumentative moves. We checked the viability of these practices in science classrooms. To do so, we investigated whether these practices led to conceptual learning, and undertook interactional analyses to study the behaviors of students and teachers. Thirty-two Grade 8 students participated in a series of activities on the day/night cycle. Learning was measured by the correctness of knowledge, the extent to which it was elaborated, the mental models that emerged from the explanations, the knowledge integration in explanations, and their simplicity. We showed that participants could learn the day/night cycle concept, as all measures of learning improved. For some students, it even led to conceptual change. However, the specific help provided by teachers during collective argumentation did not yield additional learning. The analysis of protocols of teacher-led collective argumentation indicated that although the teachers' help was needed, some teachers had difficulties monitoring these synchronous discussions. We conclude that the next step of the design-research cycle should be devoted to (a) the development of new tools directed at helping teachers facilitate synchronous collective argumentation, and to (b) activities including teachers, designers, and researchers for elaborating new strategies to use these tools to improve the already positive learning outcomes from synchronous argumentation.