Binding of Tat Signal Sequences to REMPs graph. The response curves for the wild-type signal sequences and their corresponding REMPs are shown in red. A response curve for 200 nM signal peptide-free Strep-GFP is shown in the bottom panel. doi:10.1371/journal.pone.0034159.g005 curves demonstrated that with increasing DmsD concentration, ssDmsA-GFP binding to membranes was reduced. We then tested whether DmsD can bind to membrane-bound ssDmsA-GFP. First, a solution containing 100 nM ssDmsA-GFP was injected over the immobilized membrane. ssDmsA-GFP that was not firmly bound to the membrane was washed away by Figure 6. DmsD reduces membrane binding of ssDmsA-GFP. (A) SPR response curves of various proteins injected over a phospholipid bilayer that mimics the composition of the E. coli inner membrane. Injected solutions contain either 50 nM signal peptide-free Strep-GFP (orange line), 50 nM ssDmsA-GFP (blue line), 50 nM ssDmsA-GFP with 100 nM DmsD (red line), 50 nM ssDmsA-GFP with 200 nM DmsD (green line), 50 nM TorD (grey line), or 500 nM DmsD (black line). The red and green curves were corrected for the jump in refractive index by subtracting the response curves for injections of solutions containing 100 or 200 nM DmsD, respectively. (B) SPR response curve for an experiment in which DmsD was injected over a surface consisting of ssDmsA-GFP bound to a phospholipid bilayer. ssDmsA-GFP (100 nM) was injected over an immobilized phospholipid bilayer for a period of 100 s. The surface was washed with buffer for 500 s to remove weakly bound ssDmsA-GFP. Subsequently, buffer containing 25 nM DmsD was injected. doi:10.1371/journal.pone.0034159.g006 flushing with buffer for 500 s (Figure 6B). Subsequently, buffer containing 25 nM DmsD was injected over the phospholipid membrane pre-loaded with ssDmsA-GFP. DmsD bound reversibly to this surface. Since DmsD did not bind to phospholipid bilayers alone (Figure 6A), DmsD must be able to bind ssDmsA-GFP while it is associated with the bilayer. After injection of DmsD was stopped, the DmsD dissociated rapidly, and the equilibrium level of ssDmsA- GFP bound to the membrane was restored, as highlighted by the dashed line in Figure 6B. This showed that DmsD did not recruit significant amounts of ssDmsA-GFP out of the membrane. Discussion The main question of this study was: how do REMPs discriminate between their cognate substrate(s) and other Tat substrates, which often share many similar features. We used an in vitro approach to study the interaction between REMPs and the substrate signal sequences. The key feature of this method is exposing the immobilized REMP to an intact model pre-protein in which the signal sequence of interest is fused to the N-terminus of a common cargo protein, GFP. The chimeric pre-proteins are demonstrated to be functional Tat substrates, since their expression in E. coli results in periplasmic accumulation of fluorescent, hence folded GFP [11,29]. The advantage of this approach is that it closely resembles the native situation, and at the same time rules out potential specific interactions with the mature, non-signal sequence components of the substrate. Most previous studies to probe in vitro interactions with REMPs have employed synthetic peptides that cover various parts of the substrate signal sequences [26,32]. However, the question is whether the structure and dynamics of such short synthetic peptides are comparable to the physiological situation, where the peptide is attached to a much larger protein cargo. Other studies have used intact pre- proteins [9,33], which have the disadvantage that contributions to the REMP binding from the mature domain cannot be excluded. For example, TorA from Shewanella massilia is known to have two binding sites for TorD. One of them is in the signal sequence, while the other is in the mature domain [33]. Oresnik et al. previously showed that purified recombinant DmsD interacts with the pre-protein forms of DmsA as well as TorA [9]. However, here we have shown that the signal sequences of TorA and DmsA contain all the information necessary to target the cargo protein to the correct REMP. Based on the data described here, E. coli DmsD does not bind to the signal peptide of E. coli TorA. It is thus possible that DmsD can recognize a binding site in the mature domain of TorA. The interaction between ssTorA-GFP and TorD is character- ized by Kd = 0.33 mM. The interaction between TorD and the signal sequence of TorA has been investigated previously by various techniques (Table 2). Most studies find Kd-values around 1 mM. However, one group studied the interaction between TorD and the full-length signal peptide of TorA fused at the C-terminus of the maltose-binding protein, and found a much lower Kd -value of 59 nM [32]. The influence of MBP to the affinity is unclear. The parts of the signal sequence that are important for specific REMP binding were identified using model pre-proteins with chimeric signal sequences. The signal sequences of TorA and DmsA share little sequence identity (Figure 4), though the H- regions are at least rather similar in overall composition. Also the C-region of TorA is similar in length and polarity compared that PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e34159