The Melibiose Transporter of Escherichia coli: CRITICAL CONTRIBUTION OF LYS-377 TO THE STRUCTURAL ORGANIZATION OF THE INTERACTING SUBSTRATE BINDING SITES.

We examine the role of Lys-377, the only charged residue in helix XI, on the functional mechanism of the Na(+)-sugar melibiose symporter from Escherichia coli. Intrinsic fluorescence, FRET, and Fourier transform infrared difference spectroscopy reveal that replacement of Lys-377 with either Cys, Val, Arg, or Asp disables both Na(+) and melibiose binding. On the other hand, molecular dynamics simulations extending up to 200-330 ns reveal that Lys-377 (helix XI) interacts with the anionic side chains of two of the three putative ligands for cation binding (Asp-55 and Asp-59 in helix II). When Asp-59 is protonated during the simulations, Lys-377 preferentially interacts with Asp-55. Interestingly, when a Na(+) ion is positioned in the Asp-55-Asp-59 environment, Asp-124 in helix IV (a residue essential for melibiose binding) reorients and approximates the Asp-55-Asp-59 pair, and all three acidic side chains act as Na(+) ligands. Under these conditions, the side chain of Lys-377 interacts with the carboxylic moiety of these three Asp residues. These data highlight the crucial role of the Lys-377 residue in the spatial organization of the Na(+) binding site. Finally, the analysis of the second-site revertants of K377C reveals that mutation of Ile-22 (in helix I) preserves Na(+) binding, whereas that of melibiose is largely abolished according to spectroscopic measurements. This amino acid is located in the border of the sugar-binding site and might participate in sugar binding through apolar interactions.

The substitution of Arg149 with Cys fixes the melibiose transporter in an inward-open conformation.

The melibiose transporter from Escherichia coli (MelB) can use the electrochemical energy of either H(+), Na(+) or Li(+) to transport the disaccharide melibiose to the cell interior. By using spectroscopic and biochemical methods, we have analyzed the role of Arg149 by mutagenesis. According to Fourier transform infrared difference and fluorescence spectroscopy studies, R149C, R149Q and R149K all bind substrates in proteoliposomes, where the protein is disposed inside-out. Analysis of right-side-out (RSO) and inside-out (ISO) membrane vesicles showed that the functionally active R149Q and R149K mutants could bind externally added fluorescent sugar analog in both types of vesicles. In contrast, the non-transporting R149C mutant does bind the fluorescent sugar analog as well as melibiose and Na(+) in ISO, but not in RSO vesicles. Therefore, the mutation of Arg149 into cysteine restrains the orientation of transporter to an inward-open conformation, with the inherent consequences of a) reducing the frequency of access of outer substrates to the binding sites, and b) impairing active transport. It is concluded that Arg149, most likely located in the inner (cytoplasmic) half of transmembrane helix 5, is critically involved in the reorientation mechanism of the substrate-binding site accessibility in MelB.

Hemodynamic response to exercise as measured by the solar IKG impedance cardiography module and correlation with metabolic variables.

OBJECTIVE: Impedance Cardiography (ICG) has been shown to be a feasible and accurate method for non-invasive measurement of cardiac index (CI). Aim of this investigation was the correlation of hemodynamic variables under exercise as measured by a specific ICG-monitor (Solar IKG-Modul, Version 3.0, GE-Healthcare, Freiburg, Germany) with metabolic variables. METHODS: Ten healthy volunteers were included in the investigation doing ergometer exercise (5 min equilibration followed by 5 min each at 50, 75, 100 and 125 W). Hemodynamic parameters were obtained by ICG. Metabolic variables were assessed by indirect calorimetry with the Deltatrac II Metabolic monitor using a helmet system for spontaneous respiration. RESULTS: CI increased throughout exercise (baseline: 3.0 +/- 0.4 l/min/m(2); 125 W: 4.8 +/- 0.5 l/min/m(2)). Heart rate (baseline: 87.2 +/- 13.4 bpm; 125 W: 152.7 +/- 22.4 bpm) and contractility (velocity index) (baseline: 48.9 +/- 9.3/1000 s; 125 W: 70.5 +/- 10.0/1000 s) showed a continuous rise while the stroke index decreased after an initial rise (baseline: 35.0 +/- 4.6 ml/m(2); 50 W: 37.6 +/- 4.9 ml/m(2); 75 W: 41.2 +/- 5.9 ml/m(2); 125 W: 32.3 +/- 6.1 ml/m(2)). VO(2) (baseline: 335.2 +/- 84.1 ml/min; 125 W: 1298.9 +/- 282.3 ml/min) and VCO(2)(baseline: 255.4 +/- 74.5 ml/min; 125 W: 1342.5 +/- 282.5 ml/min) increased throughout exercise. There was a good correlation in the individual fits between hemodynamic and metabolic variables. CONCLUSION: CI in healthy volunteers, as measured by the Solar IKG-Modul, correlates well with O(2)-consumption and CO(2)-production in individual subjects, thus indicating the metabolic needs under exercise conditions in healthy individuals.