Comparison of AcrB and CusA limited proteolysis.

<p>Panel A, proteolysis kinetics of CusA in C₁₂M at a 1∶1000 ratio. Panel B, proteolysis kinetics of AcrB in C₁₂M at a 1∶1000 ratio. M = Molecular weight markers.</p

Different detergents screened for CusA crystallisation.

<p>Maximal concentration obtained at the end of CusA purifications and results observed with the 192 crystallisation conditions tested for each detergent assayed. N = non-ionic detergent, Z = zwitterionic detergent.</p

Comparison of AcrB and CusA crystallisation.

<p>Panel A, AcrB crystals obtained in four conditions of initial screens in nanodrop assays. Panel B, small granules obtained with CusA in C₁₂M in initial nanodrop screens. Panel C, needles and bunches of needles obtained with CusA in C₁₂E₈. In all panels, scale bar corresponds to 100 µm.</p

Effect of various additives on CusA.

<p>Panel A, chymotrypsinolysis kinetics of CusA in C₁₂M, C₁₂E₈, and C₈FTac₅. Panel B, proteolysis kinetics of CusA in C₁₂M in the presence of different heavy metal cations. Panel C, Chymotrypsinolysis kinetics of p47phox in purification buffer alone, purification buffer with 1 mM ZnSO₄, purification buffer with 0.04% C₁₂M or purification buffer with 1 mM ZnSO₄ and 0.04% C₁₂M. Panel D, left graph corresponds to SPR measurements, dose-response double-subtracted curves of CusA in C₁₂M binding on a Ni-NTA flow cell. Increasing concentrations of CusA are: 1.4 nM, 4.1 nM, 12.3 nM, 37 nM, 111 nM, 333 nM, 1 µM and 3 µM. Right graph, chromatogram of CusA binding and elution from IMAC. Continuous line: Zn²⁺ and dashed line: Ni²⁺. FT = Flow-through, EDTA = EDTA elution.</p

Representation of proteolytic fragments of CusA projected on the AcrB structure.

<p>Panel A, the ribbon diagram and the surface of the AcrB monomer is represented in two colours: blue from the N-terminus to residue 612 (equivalent to residue 610 of CusA) and cyan from 613 to the C-terminus. Panel B, the same representation of the AcrB trimer as in panel A highlights the compacity of the region defined by residues 1 to 612 and its importance for the trimer.</p

How Do Membrane Transporters Sense pH? The Case of the Mitochondrial ADP–ATP Carrier

The activity of many membrane proteins depends markedly on the pH. Pinpointing the amino acids forming the pH sensor domains of these proteins remains challenging for current experimental techniques. Combining molecular dynamics simulations and p<i>K</i>_a predictions with in vitro transport assays, we have revealed the molecular basis of the pH dependence of the mitochondrial carrier mediating the exchanges of ADP^3– and ATP^4– across the inner mitochondrial membrane. We have demonstrated that the transport activity of this mitochondrial carrier depends on the protonation state of both the substrate and a unique, highly conserved residue of the protein. The original strategy proposed here offers a convenient framework for identifying pH-sensitive residues in membrane proteins in such cases where one single amino acid is involved. Our findings are envisioned to help toward the rational design of active compounds ranging from drugs to biosensors

Effect of the double alanine mutant in HMA6^N.

<p>Interface of helix 4 from chain A and the symmetry related chain A* with glutamate modelled at position 709 and 710, matching the WT sequence instead of the two alanine, as found in the structure. Helix 4 is shown in orange in the symmetry related side chain marked with an asterisk.</p

A matrix of RMSD values (in Å) among the N-domains of structural homologs of HMA6^N and HMA8^N compared in Fig 3 using the CA positions.

<p>A matrix of RMSD values (in Å) among the N-domains of structural homologs of HMA6^N and HMA8^N compared in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165666#pone.0165666.g003" target="_blank">Fig 3</a> using the CA positions.</p

Data collection and refinement statistics of HMA6^N-AA.

<p>Data collection and refinement statistics of HMA6^N-AA.</p

3D structures of HMA6 and HMA8 N-domains.

<p><b>A</b> The structure of HMA6^N-AA with β-strands shown in blue and α-helices in red. Missing loops are outlined by dashed lines. <b>B</b> The structure of HMA8^N; colouring is as in A. <b>C</b> Superimposition of the two structures with HMA6^N-AA in red and HMA8^N in blue.</p