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The technique of site-specific fluorescence labelling with Tetramethylrhodaminemaleimide (TMRM) in combination with two electrode voltage-clamp technique (TEVC), an approach that has been named voltage clamp fluorometry (VCF), has been used in this work to study the Na,K-ATPase. The TMRM dye has the ability to attach covalently to cysteine residues and it responds to changes in the hydrophobicity of its local environment. We exploited this property using a construct of the Na-pump in which the native, extracellularly accessible cysteines were removed and cysteine residues were introduced by site-directed mutagenesis in specific positions of the Na-pump. In this way it was possible to detect site-specific conformational rearrangements of the Na-pump in a time-resolved fashion within a native membrane environment. In particular this technique allows to resolve reactions with low electrogenicity that cannot be satisfactorily analyzed with purely electrophysiological techniques and to identify the conformations of the enzyme under specific ionic composition of the measuring buffers. We used VCF to study the influence that several cations like Na+, K+, NMG+, TEA+ and BTEA+ exert on the distribution of the Na,K-ATPase between several enzymatic intermediates and on some of the reactions related to cation transport. To this end we utilized the mutants N790C in the loop M5-M6 and the mutant E307C, T309C, L311C and E312C in the loop M3-M4. From the correspondence of the fluorescence changes with the activation and inhibition of pumping current, by K+ and ouabain respectively, and from the fact that in Na+/Na+ exchange conditions the voltage distribution of charge movement and fluorescence changes evoked by voltage jumps are in reasonable agreement we conclude that through the fluorescence signals measured from these mutants, we can indeed monitor conformational changes linked to transport activity of the enzyme. For the mutants N790 and L311, it was found that the Na+ dependence of the amplitude and kinetics of the fluorescence signal associated with the E1P-E2P transition is in agreement with the prediction of an access channel model describing the regulation of the access of extracellular Na+ to its binding site. In particular for the mutants E307 and T309 it was found that in Na+/Na+ exchange conditions, the conformational change tracked by the fluorescence was much slower than the charge relaxation at hyperpolarized potentials while the kinetics was very similar at depolarized potentials. This implies that at hyperpolarized potentials the conformational change connected to the E1P-E2P transition does not give a large contribution to the electrogenicity of the process which is also consistent with the access channel model. On the mutant N790C it was found that the external pH does not seem to have any effect on the E1P-E2P equilibrium even if it seems to modulate the fluorescence quantum yield of the dye. Fluorescence quenching experiments with iodide and D2O indicate that at hyperpolarized potentials the local environment of the mutant N790C, experiences a small change in the accessibility to water without major changes in the local electrostatic field ...
The Na+/proline transporter of E. Coli (PutP) is responsible for the uptake of proline which is subsequently used not only as a carbon and nitrogen source and a constituent of proteins but also as a particularly effective osmoprotectant. However, for a long time there was little known about the single steps in the reaction cycle of this transporter and only few details about its structure-function relationship are available. Aim of the present work was to achieve a deeper understanding about the kinetic properties of the Na+/proline transporter and to get insights into the structure-function relationship of the substrate binding. To answer these questions different techniques were used. By using the novel SSM technique combining the preparation of PutP proteoliposomes it was possible to demonstrate for the first time the electrogenic substrate binding to PutP transporter. Due to rapid solution exchange measurements on the SSM it was additionally possible to obtain time resolved information about the kinetic details of the cytoplasmic substrate binding sites which were not available by previous steady state and equilibrium binding measurements. Pre-steady-state charge translocation was observed after rapid addition of one or both of the cosubstrates Na+ and/or proline to the PutP-WT proteoliposomes adsorbed on the SSM. Thereby it was possible to link the observed electrical signals with the binding activity of PutP. The observed Na+ and/or proline induced charge displacement were assigned to an electrogenic Na+ and/or proline binding process at the cytoplasmic face of the enzyme with a rate constant of k > 50 s-1 proceeding the rate limiting step of the reaction cycle. Furthermore, based on the kinetic analysis of the electrical signals obtained from the measurements of PutP on SSM, the following characteristics of the substrates binding in PutP were deduced: (1) both Na+ and proline can bind individually to the transporter. Under physiological conditions, an ordered binding mechanism prevails; while at sufficiently high concentrations, each substrate can bind in the absence of the other; (2) substrate binding is electrogenic not only for Na+, but also for the uncharged cosubstrate proline. The charge displacement associated with Na+ binding and proline binding is of comparable size and independent of the presence of the respective cosubstrate. In addition, it was concluded that Na+ accesses its binding site through a high-field access channel resulting in a charge translocation, whereas the binding of the electroneutral proline induces a conformation alteration involving the displacement of charged amino acid residue(s) of the protein; (3) Na+ and proline binding sites interact cooperatively with each other by increasing the affinity and/or the speed of binding of the respective cosubstrate; (4) proline binding proceeds in a two step process: low affinity (~ 0.9 mM) electroneutral substrate binding followed by a nearly irreversible electrogenic conformational transition; (5) membrane impermeable PCMBS inhibits both Na+ and proline binding to the inside-out orientated PutP transporter, indicating that rather than selectively blocking a specific binding site, PCMBS probably locks the enzyme in an inactive state. The possible targets for this SH-reagent are cysteines 281 and 344 located close to the cytoplasmic surface of the protein. Beyond it, transient electrical currents of PutP were also observed on the BLM after rapid addition of proline in the presence of Na+. This was possible by combining the conventional BLM technique with high-speed flash-photolysis of caged-proline. Indeed the signals on the BLM indicate the detection of a different underlying reaction process in comparison to the data achieved by the SSM technique. This has paved the way for supplemental information about the reaction cycle since it was possible to assign the flash-photolysis BLM signals to the proline binding step followed by the internalization of Na+ and proline into the liposome. Thereby it was found, that the presence of Na+ is indispensable and the time constant for the process is ~ 63 ms. Moreover, structure-function information about the Na+ and proline binding sites of PutP was obtained by investigating the functionally important amino acid residues Asp55, Gly63 and Asp187 with site-directed mutagenesis and the combined SSM technique. One finding is that the mutated proteins PutP-D55C and PutP-G63C showed no activity on the SSM. Therefore, it can be assumed that either both Asp55 and Gly63 are crucial for the structure of PutP protein, or they are located at or close to the Na+ and proline binding sites. Furthermore, the results obtained from PutP-D187N and PutP-D187C mutants on SSM suggest that Asp187 of PutP is likely to be involved in the Na+ binding at the cytoplasmic side of the backward running carrier. Taken together the results of the present work have substantially broadened the known picture of the Na+/proline transporter PutP thereby several steps of the reaction cycle were elucidated, and moreover, valuable insights into the structure-function relationship of the transporter have become available.
Group III presynaptic metabotropic glutamate receptors (mGluRs) play a central role in regulating presynaptic activity through G-protein effects on ion channels and signal transducing enzymes. Like all Class C G-protein coupled receptors, mGluR8 has an extended intracellular C-terminal domain (CTD) presumed to allow for modulation of downstream signaling. To elucidate the function and modulation of mGluR8, yeast two-hybrid screens of an adult rat brain cDNA library were performed with the CTDs of mGluR8a and 8b (mGluR8-C) as baits. Different components of the sumoylation cascade (ube2a, sumo-1, Pias1, Pias gamma and Pias xbeta) and some other proteins were identified as mGluR8 interacting proteins. Binding assays using recombinant GST-fusion proteins confirmed that Pias1 interacts not only with mGluR8-C, but all group III mGluR CTDs. Pias1 binding to mGluR8-C required a region N-terminally to a consensus sumoylation motif and was not affected by arginine substitution of the conserved lysine K882 within this motif. Co-transfection of fluorescently tagged mGluR8a-C, sumo-1 and enzymes of the sumoylation cascade into HEK 293 cells showed that mGluR8a-C can be sumoylated in cells. Arginine substitution of lysine K882 within the consensus sumoylation motif, but not of other conserved lysines within the CTD, abolished in vivo sumoylation. The results are consistent with post-translational sumoylation providing a novel mechanism of group III mGluR regulation.
The melibiose permease (MelB) of E.coli functions as a secondary-active symporter by using the electrochemical H+, Na+, or Li+ gradient to accumulate, e.g., melibiose [review in Pourcher et al. 1990a]. The global and primary objective of this thesis was to apply pre-steady state methods for the investigation of reaction rates of individual steps in the cycle of MelB. Especially the melibiose binding induced transition was investigated by the solid-supported membrane (SSM) technique [Seifert et al. 1993] in combination with a rapid solution exchange system [Pintchovius and Fendler 1999] and with the Stopped-flow technique [Roughton 1934]. To approach this goal, either wild-type or mutated MelB were purified and reconstituted into liposomes as described [Pourcher et al. 1995]. Although the orientation of the proteins is a critical factor for the activity of MelB, it was, so far, unknown. To determine the orientation of the proteins in the liposomes, single Cys mutants R139C and R141C [Abdel-Dayem et al. 2003] were selectively labeled with 3-(N-maleimidylpropionyl)biocytin (MPB) and analyzed by SDS-PAGE and Western Blot. The assay indicated that most of the proteins are inside-out (ISO) oriented permitting to relate the pre-steady state electrical and fluorescence signals to the reverse transport activity of MelB. The melibiose induced electrical signal was investigated in wild-type MelB with the SSM technique. The transporter was activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na+ at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na+ binding to the transporter, nor to the displacement of already bound Na+ within MelB. Electrogenic melibiose binding is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na+ and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. Melibiose binding is fast in the presence of Na+ (k > 50 s-1). Furthermore, two previously identified transport deficient mutants of loop 4-5, R141C and E142C [Abdel-Dayem et al. 2002, Séry 2002], were purified and extensively studied with the SSM. Whereas the electrical signals from control cysteine-less mutant showed a bi-exponential time course of decay, those from R141C or E142C consisted of only a single fast exponential component, and the slow decaying component associated with substrate translocation was missing. The electrical signals evoked by a melibiose concentration jump in the presence of Na+ were much smaller than the corresponding signals in C-less MelB. Furthermore, R141C lost the stimulating effect of melibiose on Na+ binding. Steady-state Trp fluorescence spectroscopy revealed impaired conformational changes after melibiose binding in the mutants and fluorescence resonance energy transfer (FRET) measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that loop 4-5 contributes to the coordinated interactions between the ion- and sugar binding site and participates in conformational changes after melibiose binding that are essential for the subsequent obligatory coupled translocation of substrates. By using the Stopped-flow technique, three different approaches were followed. First, the intrinsic Trp fluorescence of MelB, known to increase upon melibiose binding [Mus-Veteau et al. 1995], revealed a signal with a T 1 of ~15 ms in C-less. This time constant is of the same order of magnitude as that determined with the SSM method suggesting that Trp fluorescence and electrical signal are related processes. Conformation for this assumption came from the fact that the activation energies Ea for both processes are similar (around 45 KJ/mol). Second, by using the fluorescent sugar analog Dns2-S-Gal, which monitors events close to the sugar binding site [Maehrel et al. 1998], a signal with a T 1 of ~18 ms was recorded upon Na+ addition. Finally, the fluorescent dye MIANS was used to selectively label the single Cys mutant E365C of loop 10-11. Stopped-flow measurements revealed a melibiose-induced fluorescent signal with a T 1 of 45 ms. Since electrical measurements with the MIANS-labeled E365C excluded the possibility that the label is responsible for the slower kinetics, the conformational change detected by the MIANS fluorescence was assigned to a slow transition in the cycle of MelB after melibiose binding. Ea was determined to be 96 KJ/mol corroborating, thus, the hypothesis of a different process. In conclusion, it was possible to correlate the electrical and fluorescence signals to partial reactions of the transport cycle and to determine their rate constants. According to this new model, the melibiose-induced signal detected with the Trp and electrical measurements corresponds to a step preceding the carriers’ reorientation (3 <-> 3*, k ~ 65s-1), and the melibiose-induced signal detected with the MIANS fluorescence to the reorientation itself (3* <-> 4, k ~ 20s-1).