Binding of Tat Signal Sequences to REMPs Figure 3. Twin-arginines are not essential for REMP binding. (A) SPR response curves obtained by injecting solutions containing 100 nM ssTorA-GFP (black line) or 100 nM ssTorA(KKK)-GFP (red line) over immobilized TorD. (B) Response curves obtained by injecting solutions of 100 nM ssDmsA-GFP (black line) or 100 nM ssDmsA(KK)-GFP (red line) over immobilized DmsD. doi:10.1371/journal.pone.0034159.g003 regions were delineated according to the scheme shown in Figure 4. This exercise resulted in a total of eight chimera pre- protein constructs (Figure 5). These pre-proteins containing region-swapped signal peptides were injected over immobilized TorD and DmsD at different concentrations, ranging from 20 to 400 nM, of which the response curves at 200 nM are shown for all pairings in Figure 5. The main purpose of this qualitative comparison was to establish in a qualitative manner whether there is an interaction between various chimeric signal peptides and REMPs. In this experimental set, we used strep-tagged GFP lacking any signal peptide as negative control and the GFPs with wild type signal peptides (named TTT-GFP and DDD-GFP in Figure 5, respectively) as positive controls for binding. In case of ssTorA, the only chimeric signal sequence protein that bound with reasonable affinity to TorD was the DTT-GFP fusion, in which the N-region was replaced for the one of DmsA. When either the H- or the C-region of ssTorA was replaced, binding was hardly detected even at the highest pre-protein concentration, indicating that the binding site for TorD was lost. These TorA-based chimeric pre-proteins were also tested for binding to DmsD. However, none of these TorA-based signal sequences displayed any binding to DmsD (Figure 5). For ssDmsA, similar results were observed. Its N-region could be replaced without affecting DmsD binding, but when either the H- or the C-region was replaced, binding to DmsD could hardly be detected. Apparently, the combined H- and the C-regions but not the N-region of ssTorA and ssDmsA are essential for binding to the corresponding REMP. DmsD reduces membrane binding of ssDmsA-GFP The second aim of this work was to find out how REMP-signal sequence binding relates to the next step in translocation. In earlier work, we showed that signal peptides mediate binding of Tat substrates to phospholipid bilayers and biological membranes, and speculated that this binding might be a step en-route to translocation [11]. Furthermore, it has been hypothesized that the role of REMP binding to signal peptides is to prevent the premature interaction of incomplete or unfolded substrates with the Tat translocase [23]. Accordingly, it would be interesting to investigate membrane binding of pre-proteins in the presence and absence of REMPs. To this end, phospholipid bilayers mimicking the E. coli inner membrane lipid composition were immobilized on an L1 SPR chip. First we tested whether the REMPs themselves could bind to these membrane surfaces. No interaction of DmsD alone with membranes was found (Figure 6A), which is in agreement with earlier work [16]. The step-like SPR response seen for DmsD solely reflects a change in refractive index of the injected solution. In contrast, TorD could bind to phospholipid bilayers directly (Figure 6A), and therefore the TorD-ssTorA pair was not used in this experiment. A solution containing 50 nM ssDmsA- GFP was injected over a freshly made membrane surface (Figure 6A). ssDmsA-GFP did bind to the bilayer, as reported previously [11]. Membrane binding is caused by the signal peptide, since signal peptide-free Strep-tagged GFP did not bind to the membrane (Figure 6A). Subsequently, 50 nM ssDmsA-GFP was co-injected with 100 or 200 nM of DmsD. The resulting SPR Figure 4. Alignment of DmsA and TorA signal sequences. The signal sequences of the DmsD substrates DmsA, YnfF and YnfE were aligned using ClustalW. Similarity of residues for these three sequence is summarized in the fourth row (* = identical, : = strong similarity, N = weak similarity). For comparison the signal sequence of TorA is shown in the bottom row. Regions of the TorA sequence that show remarkable similarity with the consensus sequence of DmsD substrates are indicated in grey boxes. N-regions are shown in black type, H-regions in red and C-regions blue, and the two arginines of the twin-arginine motif are typed in bold. doi:10.1371/journal.pone.0034159.g004 PLoS ONE | www.plosone.org 6 March 2012 | Volume 7 | Issue 3 | e34159