LRET

Tb³⁺ acted as LRET donor in our experiments. It was embedded in a high affinity (K_d = 57 ± 3 nM) lanthanide binding tag that consisted of the following 17 amino acids: YIDTNNDGWYEGDELLA.²⁷ Its tryptophan served as antenna for efficient Tb³⁺excitation at 266 nm by a Nd:YAG-laser system (VM-TIM, Jena, Germany). We used a home-built LRET setup as described.²⁸ Briefly, the laser light passed a dichroitic mirror (z266rdc, Chroma, Bellows Falls, VT, USA) and was focused on the sample via a 40 × 1.25 glycerol immersion quartz objective (Sysmex Partec GmbH, Münster, Germany). The objective also served to collect the light emitted by Tb³⁺ or the acceptor molecules, Atto488 or Atto532. These fluorescent dyes were attached via maleimide linkers to genetically introduced cysteines in SecA. Both Tb³⁺ luminescence and the fluorescent light further went through the dichroic mirror to a longpass filter (HQ465lp, Chroma, Bellows Falls, VT, USA), which removed residual excitation light. Close proximity between Tb³⁺ and the fluorescent dye resulted in a decrease of luminescence lifetime. Alternatively, to donor luminescence, we measured acceptor fluorescence (sensitized emission). For that purpose, a bandpass filter (D520/25 m or HQ567/15×, Chroma, Bellows Falls, VT, USA) was additionally inserted into the emission beampath. In both measurement modes the light was absorbed by a gated single photon avalanche diode (SPCM-AQRH, Perkin Elmer, Waltham, MA, USA), connected to a DSN-101 power supply (PicoQuant, Berlin, Gremany). The signal was recorded and triggered by a multichannel scaler card (NanoHarp 250, PicoQuant, Berlin, Gremany). A delay of 30 μs between the onset of the light pulse (5–6 ns pulse duration) and diode activation helped to avoid any interference of the measurement with direct acceptor excitation.

As a system check, we used parts of the two-helix-finger, the C-terminal α-helix of SecA, as a molecular ruler. We measured a distance of 47 Å for the unbound SecA K797C-Atto488 V829LBT double mutant, which perfectly agrees with estimates from crystal structures (2fsi to 47.5 Å, 2fsh to 47.1 Å, 2ipc to 49.7 Å).