Binding of Tat Signal Sequences to REMPs Figure 1. The signal sequence ensures specific REMP binding. Surface plasmon resonance sensorgrams for the injection of various proteins over immobilized TorD (A) and DmsD (B) are shown. The model pre-proteins ssDmsA-GFP (black lines), ssTorA-GFP (red lines) and signal sequence- free GFP (blue lines) were injected for 60 seconds at a concentration of 200 nM, a flow rate of 50 ml/min, and a temperature of 25uC. doi:10.1371/journal.pone.0034159.g001 are shown in Figure 2B. A small drop is observed during the injection phase of the SPR response curves at the highest pre- protein concentrations, approximately 15 seconds after injection. This drop is hardly present in the raw data (Figure 2A), and thus results from the correction procedure. This is probably caused by the following. An unliganded Ni-NTA surface has more Ni2+ ions available for unspecific binding to ssTorA-GFP than a surface to which TorD is bound. As a result, subtraction of the control curve results in an over-correction for unspecific binding, which leads to the observed drop in the SPR intensity. Fortunately, the effect is small and mainly seen at the highest pre-protein concentrations. We therefore continued to quantify the corrected SPR curves. The SPR intensity at equilibrium (Ieq) was determined as the average of the signal intensity over the shaded region in Figure 2A at each pre-protein concentration. The data are represented in a Scatchard plot, in which Ieq divided by the concentration of pre- protein is plotted against Ieq (Figure 2C). A linear function describes the data well, which is a strong indication of equimolar binding. Ieq was then plotted against the concentration of pre- protein (Figure 2D), and fitted using a Langmuir model for 1:1 binding Ieq~ Ieq,max:C CzKd 1 ð Þ Ieq,max is the equilibrium SPR intensity at a saturating pre-protein concentration, C is the concentration of pre-protein and Kd is the dissociation constant of the pre-protein - REMP complex. The resulting Kd for TorD:ssTorA-GFP is 0.3360.04 mM (Table 2). The same approach was followed for the binding of ssDmsA- GFP to DmsD. However, in this case the Scatchard plot was non- linear, which indicated that the binding of ssDmsA-GFP to DmsD did not follow a 1:1 model. Hence the Langmuir equation does not apply, and the Kd-value could not be reliably determined. The role of the twin-arginine motif in pre-protein binding to REMPs To determine whether the double arginine motif in the TorA signal peptide has any role in pre-protein binding to TorD, either one or both of the twin-arginine motif residues (RR) was mutated to lysine, resulting in ssTorA(RK)-GFP, ssTorA(KR)-GFP, and ssTorA(KK)-GFP. These mutations are known to affect translo- cation: ssTorA(RK)-GFP and ssTorA(KR)-GFP show strongly reduced translocation efficiencies, whereas ssTorA(KK)-GFP is not transported at all [24,25]. The mutant pre-proteins were purified and their affinity for TorD was measured by SPR (Figure 2 D) and quantified using Equation 1. All three mutant pre-proteins bind approximately ten-fold more weakly compared to the wild- type ssTorA-GFP (Table 2). In the signal sequence of TorA, the residue following the two conserved arginine residues happens to be an arginine as well. In order to rule out that the third arginine takes over any role of the two conserved ones in the binding to TorD, this residue was also mutated to a lysine, resulting in ssTorA(KKK)-GFP. The dissociation constant of 4.9 mM found for ssTorA(KKK)-GFP binding to TorD is similar to that obtained for ssTorA(KR)-GFP and ssTorA(RK)-GFP, and approximately ten-fold higher than for the wild-type signal sequence (Figure 3A, Table 2). Moreover, a more drastic mutation of signal sequence was made in which the three arginine residues were changed to the tripeptide glycine- histidine-proline (ssTorA(GHP)-GFP). This pre-protein was ex- pressed, purified and tested for binding to TorD. However, no binding could be detected (data not shown). The two arginines in the signal peptide of DmsA were also mutated to lysine. The SPR curve for a 100 nM solution of the pre-protein ssDmsA(KK)-GFP binding to DmsD was qualitatively very similar to that for wild-type ssDmsA-GFP at the same concentration (Figure 3B). Moreover the SPR intensities at equilibrium were very similar for both pre-proteins (not shown). For ssDmsA(KK)-GFP, binding of the mutant signal sequence to DmsD could not be readily quantified because the interaction does not follow the 1:1 binding model. Nevertheless, it can be concluded that conservative mutation of the RR-motif to lysine(s) has very little effect on ssDmsA-GFP binding to DmsD. In conclusion, the conserved arginine pair in the signal peptides of the Tat substrates TorA and DmsA does not absolutely determine binding of the peptides to the cognate REMP. Conservative mutations to lysine residues are tolerated. However, for ssTorA binding to TorD more drastic mutations in which the positively charged residues are removed result in loss of binding. Specific binding depends on H- and C-regions The amino acid sequences of the signal peptides of TorA and DmsA were compared in order to assess which part(s) of the signal PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e34159