A double-Cys variant of the 101-amino-acid-residue protein S6 from Thermus thermophilus was constructed in which Met1 and Phe97 were replaced with Cys. The DNA sequence of the S6 gene (constructed using Escherichia coli-optimised codons) was custom synthesised (GenScript) and the fragment cloned into a pET28a expression vector using NcoI/XhoI restriction sites. This cloning resulted in an open reading frame without any affinity tag and the sequence Met-Gly placed immediately before Cys1. The protein was expressed in E. coli BL21 (DE3) and purified by anion-exchange chromatography followed by size-exclusion chromatography to >95% purity as described.17 (link) For smFRET experiments, the protein was site-specifically labelled via thiol–maleimide chemistry with maleimide-functionalised FRET donor (ATTO 532, Atto-Tec) and acceptor (Abberior STAR 635P, Abberior) fluorophores, following standard procedures. The labelled protein was separated from unbound dyes by size-exclusion chromatography.
Atto 532
Manufactured by ATTO-TEC
Sourced in Germany
The ATTO 532 is a fluorescent dye with an excitation maximum at 532 nm and an emission maximum at 553 nm. It is designed for use in fluorescence-based applications.
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Lab products found in correlation
Atto647n, by ATTO-TEC (1 mentions)
Molecular biology grade water, by Thermo Fisher Scientific (1 mentions)
Adenosine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Cytidine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Guanosine 5 monophosphate disodium salt, by Merck Group (1 mentions)
Star 635p, by Abberior (1 mentions)
Atto 647, by ATTO-TEC (1 mentions)
5 protocols using atto 532
1
Single-Molecule FRET Labeling of S6 Protein
2
CFTR Hairpin Variants for FRET
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CFTR wild-type and V232D mutant TM3/4 hairpin variants for site-specific double labeling were constructed with two Cys residues placed at the N- and C-terminal ends of the CFTR sequence (see Supplementary Fig. 5 ). Hairpins were produced and purified as previously described9 (link),37 (link),38 with minor modifications as detailed in Supplementary Information (see also Supplementary Fig. 9 ). Labeling with FRET donor (ATTO532; Atto-Tec, Siegen, Germany) and acceptor (ATTO647N; Atto-Tec) fluorophores was performed following published procedures39 (link),40 (link) as described in Supplementary Information . Hairpins were reconstituted into large unilamellar vesicles (LUVs) (see also Supplementary Fig. 10 and Supplementary Table 3 ) to yield proteoliposomes with a protein-to-vesicle molar ratio of <1:10 (i.e., less than every tenth LUV contained one hairpin molecule). Details on hairpin design, production, purification, labeling, LUV preparation, and hairpin reconstitution are given in Supplementary Information .
3
Fabrication of Microfluidic Devices
4
Expression and Purification of OmpX Variants
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A tag-free double-cysteine variant of OmpX (OmpX1,149) without a signal sequence was expressed as inclusion bodies in Escherichia coli BL21(DE3) cells and purified following standard procedures including anion exchange chromatography, as previously reported (69 (link), 70 (link)). After refolding, the protein was labeled with FRET donor (ATTO532, Atto-Tec) and acceptor (Abberior STAR 635P, Abberior) dyes. We further produced two other OmpX variants; however, they were not used for the main study due to their misbehavior possibly caused by higher propensity toward aggregation (SI Appendix, Fig. S8 ). Details are given in SI Appendix .
Skp and SurA were produced as N-terminal hexahistidine (His6) fusion proteins in E. coli BL21 (DE3) cells and purified using immobilized metal affinity chromatography under denaturing conditions, as previously reported (32 (link), 59 (link)). The proteins were refolded prior to experiments. Details on the production and purification are given inSI Appendix .
Skp and SurA were produced as N-terminal hexahistidine (His6) fusion proteins in E. coli BL21 (DE3) cells and purified using immobilized metal affinity chromatography under denaturing conditions, as previously reported (32 (link), 59 (link)). The proteins were refolded prior to experiments. Details on the production and purification are given in
5
Labeling Sti1 Proteins for spFRET
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For spFRET experiments, native cysteines were removed and different double-cysteine mutants were generated such that the donor and acceptor fluorophores could be labelled at specific locations. The Sti1 double-cysteine variants were labelled stochastically at the cysteines with ATTO 532 and ATTO 647 (AttoTec) with a threefold excess of labels in 40 mM Hepes (pH 7.5), 150 mM KCl, 5 mM MgCl2 for 1 h at room temperature. The reaction was quenched with a 10-fold excess of DTT, and free label was separated from the protein on a Superdex 200 10/300 GL HPLC column (GE Healthcare). The cysteine mutations and fluorescent labelling did not alter the functionality of Sti1 (Supplementary Fig. 1d ).
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