BasC smFRET experiments
Description
Raw data to make the figures of the article: The conserved lysine residue in transmembrane helix 5 is key for the cytoplasmic gating of the L-amino acid transporters Authors: Joana Fort*, Adrià Nicolàs-Aragó, Luca Maggi, Maria Martinez Molledo, Despoina Kapiki, Paula Gonzalez-Novoa, Patricia Gómez-Gejo, Niels Zijlstra, Susanna Bodoy, Els Pardon, Jan Steyaert, Oscar Llorca, Modesto Orozco, Thorben Cordes,*, Manuel Palacín* * corresponding Authors To monitor the movement of the inner gate of BasC in solution at room temperature, we developed a smFRET assay that tracks its conformational changes. To this end, two distinct double-cysteine variants of BasC were designed. Ile7 in TM1a was combined with two other residues in the protein: Thr120 in TM4, forming the TM1a-TM4 pair, and Cys427, the only natural cysteine in BasC, in TM12, forming the TM1a-TM12 pair (Fig. 1A). ALEX-smFRET microscopy All samples were examined by focusing the excitation/observation volume of a confocal microscope into a ~100 μl sample drop containing ~50 pM of BasC in DDM buffer on a glass coverslip. Before data recording, the coverslips were coated for >60 s with 1 mg/mL bovine serum albumin to prevent fluorophore and/or protein adsorption. Each experimental condition (apo, ligand etc.) was then studied for between 30 and 120 min, depending on the fraction of donor- and acceptor-containing BasC molecules in relation to donor- and acceptor-only ones. We also performed DNA controls with the same fluorophore pairs as for the proteins in DDM buffer to verify proper microscope alignment and to exclude buffer interferences. For alternating laser excitation, we used two laser sources for sample excitation with electronic modulation: a 532 nm diode laser at 60 μW (OBIS 532-100-LS, Coherent, USA) and a diode laser at 640 nm (OBIS 640-100-LX, Coherent, USA) at 25 or 30 μW for Alexa 546-Alexa 647 and sCy3-sCy5 dye-pairs, respectively. The laser light was guided into an epi-illuminated confocal microscope body (Olympus IX71, Hamburg, Germany) by a dual-edge beamsplitter (ZT532/640rpc, Chroma/AHF, Germany) and focused to a diffraction-limited excitation spot by a water immersion objective (UPlanSApo 60x/1.2w, Olympus Hamburg, Germany). The emitted fluorescence was collected through the same objective, spatially filtered using a pinhole with a diameter of 50 μm and spectrally split into donor and acceptor channel by a single-edge dichroic mirror H643 LPXR (AHF). Fluorescence emission was filtered (donor: BrightLine HC 582/75 (Semrock/AHF), acceptor: Longpass 647 LP Edge Basic (Semroch/AHF)) and focused onto avalanche photodiodes (SPCM-AQRH-64, Excelitas). The detector outputs were recorded by a NI-Card (PCI-6602, National Instruments, USA). data