Binding of Tat Signal Sequences to REMPs Figure 2. Determination of the dissociation constant for ssTorA-GFP binding to immobilized TorD. (A) Raw SPR sensorgrams and (B) referenced sensorgrams of ssTorA-GFP binding to immobilized TorD at the following pre-protein concentrations (shown from dark to light grey): 39 nM, 78 nM, 156 nM, 313 nM, 625 nM, 1250 nM. The sensorgram for a buffer injection is shown in black. (C) Equilibrium SPR intensity for binding of ssTorA-GFP to TorD at various concentrations in a Scatchard plot (open symbols, see main text for details). The intensity values used are the average SPR response derived from the shaded area (22–28 s) in panel A. The data are fitted to a straight line (black). (D) The SPR intensity at equilibrium is plotted as a function of pre-protein concentration, for binding to immobilized TorD. The pre-proteins used are ssTorA-GFP (shown in black), ssTorA(RK)-GFP (shown in blue), ssTorA(KR)-GFP (shown in green) and ssTorA(KK)-GFP (shown in red). The best fit of the Langmuir binding isotherm (Equation 1) to the data is shown as a line in the corresponding color. doi:10.1371/journal.pone.0034159.g002 1Dissociation constants from this work are obtained from SPR data by fitting the Langmuir equation (Equation 1) to the equilibrium SPR response as a function of pre-protein concentration. Errors are standard fitting errors. 2ss, signal sequence; GFP, green fluorescent protein; MBP, maltose binding protein; SBP, streptavidin binding peptide. doi:10.1371/journal.pone.0034159.t002 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e34159 Table 2. Dissociation constants for TorA signal peptides binding to TorD. Signal sequence Kd (mM)1 Method Construct2 TorA TorA(KR) TorA(RK) TorA(KK) TorA(KKK) TorA TorA TorA 0.3360.04 SPR 0.5460.04 SPR 2.960.1 3.660.2 4.960.1 1.7 0.059 3.8 SPR SPR SPR ITC ITC SPR ss-GFP ss-GFP ss-GFP ss-GFP ss-GFP MBP-ss ss-SBP peptide S10-R26 Reference this work this work this work this work this work [26] [32] [19] sequence are responsible for the specific interaction with the corresponding REMP. The signal sequences of YnfE and YnfF were included in this exercise, since these paralogs of DmsA are also known substrates of DmsD [10,31]. First, the sequences of DmsA, YnfE and YnfF were aligned together using ClustalW (Figure 4). Apart from the consensus twin-arginine motif S/ TRRxFLK that is found in all Tat substrates, the signal sequences of the DmsD substrates are remarkably similar in both the H- region and the beginning of the C-region. Second, the signal sequence of TorA was included in the alignment. Despite a similar overall composition, the signal sequence of TorA does not align so well with the other three signal sequences without the introduction of insertions and deletions (Figure 4). The high similarity of the DmsD substrates in their H- and C- regions and the lack of similarity in the same regions of the TorA signal suggest that these regions might be important for specific recognition by the correct REMP. The validity of this hypothesis was tested by creating region-swapped chimeric signal sequences, in which either the N- or the H- or the C-regions of ssTorA-GFP and ssDmsA-GFP were replaced by the corresponding region from the other signal sequence. The borders between the different