tetramethylrhodamine and fluorescein-5-maleimide

The Na+/K+-ATPase maintains the physiological Na+ and K+ gradients across the plasma membrane in most animal cells. The functional unit of the ion pump is comprised of two mandatory subunits including the alpha-subunit, which mediates ATP hydrolysis and ion translocation, as well as the beta-subunit, which acts as a chaperone to promote proper membrane insertion and trafficking in the plasma membrane. To examine the conformational dynamics between the alpha- and beta-subunits of the Na+/K+-ATPase during ion transport, we have used fluorescence resonance energy transfer, under voltage clamp conditions on Xenopus laevis oocytes, to differentiate between two models that have been proposed for the relative orientation of the alpha- and beta-subunits. These experiments were performed by measuring the time constant of irreversible donor fluorophore destruction with fluorescein-5-maleimide as the donor fluorophore and in the presence or absence of tetramethylrhodamine-6-maleimide as the acceptor fluorophore following labeling on the M3-M4 or M5-M6 loop of the alpha-subunit and the beta-subunit. We have also used fluorescence resonance energy transfer to investigate the relative movement between the two subunits as the ion pump shuttles between the two main conformational states (E1 and E2) as described by the Albers-Post scheme. The results from this study have identified a model for the orientation of the beta-subunit in relation to the alpha-subunit and suggest that the alpha- and beta-subunits move toward each other during the E2 to E1 conformational transition.

Topics: Animals; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Hydrolysis; Ions; Microscopy, Fluorescence; Oocytes; Patch-Clamp Techniques; Protein Conformation; Protein Structure, Secondary; Rhodamines; Sheep; Sodium-Potassium-Exchanging ATPase; Xenopus laevis

The aspartate receptor of bacterial chemotaxis is representative of a large family of taxis receptors widespread in prokaryotes. The homodimeric receptor associates with cytoplasmic components to form a receptor-kinase signaling complex. Within this complex the receptor is known to directly contact the histidine kinase CheA, the coupling protein CheW, and other receptor dimers. However, the locations and extents of the contact regions on the receptor surface remain ambiguous. The present study applies the protein-interactions-by-cysteine-modification (PICM) method to map out surfaces on the aspartate receptor that are essential for kinase stimulation in the assembled receptor-kinase complex. The approach utilizes 52 engineered cysteine positions scattered over the surface of the receptor periplasmic and cytoplasmic domains. When the bulky, anionic probe 5-fluorescein-maleimide is coupled to these positions, large effects on receptor-mediated kinase stimulation are observed at eight cytoplasmic locations. By contrast, no large effects are observed for probe attachment at exposed positions in the periplasmic domain. The results indicate that essential receptor surface regions are located near the hairpin turn at the distal end of the cytoplasmic domain and in the cytoplasmic adaptation site region. These surface regions include the docking sites for CheA, CheW, and other receptor dimers, as well as surfaces that transmit information from the receptor adaptation sites to the kinase. Smaller effects observed in the cytoplasmic linker or HAMP region suggest this region may also play a role in kinase regulation. A comparison of the activity perturbations caused by a dianionic, bulky probe (5-fluorescein-maleimide), a zwitterionic, bulky probe (5-tetramethyl-rhodamine-maleimide), and a nonionic, smaller probe (N-ethyl-maleimide) reveals the roles of probe size and charge in generating the observed effects on kinase activity. Overall, the results indicate that interactions between the periplasmic domains of different receptor dimers are not required for kinase activation in the signaling complex. By contrast, the observed spatial distribution of protein contact surfaces on the cytoplasmic domain is consistent with both (i) distinct docking sites for cytoplasmic proteins and (ii) interactions between the cytoplasmic domains of different dimers to form a trimer-of-dimers.

Topics: Aspartic Acid; Chemotaxis; Cysteine; Cytoplasm; Dimerization; Enzyme Activation; Ethylmaleimide; Fluoresceins; Fluorescent Dyes; Models, Molecular; Mutagenesis, Site-Directed; Peptide Library; Protein Interaction Mapping; Protein Kinases; Protein Structure, Tertiary; Receptors, Amino Acid; Rhodamines; Salmonella typhimurium; Surface Properties