Binding of Tat Signal Sequences to REMPs Table 1. List of plasmids used in the study. Plasmid pTYB11-TorA-GFP pTYB11-TorA(KR)-GFP pTYB11-TorA(RK)-GFP pTYB11-TorA(KK)-GFP pTYB11-TorA-KKK-GFP pTYB11-DmsA-GFP pTYB11-DmsA(KK)-GFP pTYB11-TTD-GFP pTYB11-TDT-GFP pTYB11-DTT-GFP pTYB11-DDT-GFP pTYB11-DTD-GFP pTYB11-TDD-GFP pET16b-TorD pET16b-DmsD pGFP6A Reference [11] This work This work This work This work [11] This work This work This work This work This work This work This work This work This work [38] Protein of interest ssTorA-GFP with self cleavable intein tag ssTorA(KR)-GFP with self cleavable intein tag ssTorA(RK)-GFP with self cleavable intein tag ssTorA(KK)-GFP with self cleavable intein tag ssTorA(KKK)-GFP with self cleavable intein tag ssDmsA-GFP with self cleavable intein tag ssDmsA(KK)-GFP with self cleavable intein tag ssTorA-GFP with self cleavable intein tag, with the C-region of ssDmsA ssTorA-GFP with self cleavable intein tag, with the H-region of ssDmsA ssTorA-GFP with self cleavable intein tag, with the N-region of ssDmsA ssDmsA-GFP with self cleavable intein tag, with the C-region of ssTorA ssDmsA-GFP with self cleavable intein tag, with the H-region of ssTorA ssDmsA-GFP with self cleavable intein tag, with the N-region of ssTorA TorD with N-terminal His-tag DmsD with N-terminal His-tag Strep-GFP doi:10.1371/journal.pone.0034159.t001 SPR measurements Surface plasmon resonance measurements were performed on Biacore 2000 or Biacore T100 systems (Biacore AB, Uppsala, Sweden) at 25uC and a flow rate of 50 ml/min, unless indicated otherwise. All solutions were freshly prepared, degassed, and filtered through membranes with 0.22 mm pores. Nitriloacetic acid (NTA) chips providing affinity for Ni-ions were used to study interaction between pre-proteins and REMPs. The running buffer was 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 50 mM ethylenediaminetetraacetic acid (EDTA), and 0.005% P20 detergent at pH 7.4. In each cycle, 50 ml of 0.5 mM NiCl2 in running buffer was injected to activate the surface, followed by injection of His-tagged REMP in running buffer, except for the reference surface, to which no protein was bound. For qualitative binding studies, REMPs were immobilized to a readout of 1000 response units (RU) in order to maximize the sensitivity of the experiment. For quantitative experiments in which dissociation constants were determined, a lower level of immobilization (500 RU) was employed, to minimize non-specific binding of the pre-proteins to the reference surface. The concentration of pre-proteins was varied between 10 and 6400 nM. After each interaction measurement, the SPR chip was regenerated with 250 mM EDTA. L1 sensor chips (Biacore AB) on which lipophilic groups are covalently attached to the surface via a dextran matrix were used to study protein interactions with membranes. Small unilamellar vesicles (SUVs) were generated from E. coli membranes or from lipids mimicking the E. coli inner membrane composition as described in our previous work [11]. SUVs were spread and immobilized on the sensor chip [11]. After completion of an experiment, the surface of the chip was regenerated by washing with 40 mM N-octyl -D-glucopyranoside. Sequence alignment The signal sequences of TorA (residues 1–42), DmsA (residues 1–45), YnfE (residues 1–43) and YnfF (residues 1–49) were aligned using the online tool ClustalW (version 1.83, http://www.ch. embnet.org/cgi-bin/clustalw_parser) with default parameters: open gap penalty 10, extending gap penalty 0.05, an end gap penalty 10, and a separation gap penalty 0.05. Results Specific interaction of REMPs with chimeric Tat pre- proteins Binding of the Tat signal peptides to REMPs was investigated by SPR. His-tagged DmsD and TorD were immobilized in separate lanes on the surface of a Ni-NTA chip. The signal sequence of DmsA fused to GFP (ssDmsA-GFP) and the signal sequence of TorA fused to GFP (ssTorA-GFP) were separately injected over this surface for a time period of 60 s. The observed SPR responses (Figure 1) imply that ssDmsA-GFP binds to the DmsD covered surface, but not to the TorD surface. Similarly ssTorA-GFP shows specific binding to TorD. GFP by itself did not bind to either TorD or DmsD (Figure 1); the step-like SPR responses observed are a direct result of the higher refractive index of the protein solution compared to the protein-free buffer. The specific interactions between the REMPs and their cognate GFP- fusion pre-proteins thus are apparently governed by the respective Tat signal peptide. Affinity constants for the interaction between REMPs and pre-proteins The affinity of TorD for the signal sequence of TorA was quantified by recording SPR response curves at different pre- protein concentrations (Figure 2A). A low flow rate of 20 ml/min was used to increase the contact time and hence allow the binding reaction to equilibrate.. To test whether non-specific binding of ssTorA-GFP directly to the Ni-NTA surface contributes to the SPR curves, the same pre-protein solutions were also injected over a reference surface to which no TorD was immobilized. Since binding of ssTorA-GFP to this reference surface was indeed observed, the raw SPR curves were corrected for non-specific binding by subtracting the reference curves. The resulting curves PLoS ONE | www.plosone.org 3 March 2012 | Volume 7 | Issue 3 | e34159