Quartz glass cuvette

The emission spectra of the NPs in hexane and the emission spectra of the NP-stained PSMPs in ethanol were recorded with a calibrated FSP920 fluorescence spectrometer from Edinburgh Instruments Ltd. at RT ((25 ± 2) °C) using (10 × 10) mm quartz glass cuvettes (Hellma GmbH). Excitation was always at 350 nm.
Measurements of the PL decay curves of these samples, the fits of which providing the respective fluorescence lifetimes (FLTs), as described in the SI (see Eqs. SE1 and SE2) were performed with a calibrated FLS920 fluorescence spectrometer from Edinburgh Instruments Ltd. in (10 × 10) mm quartz glass cuvettes (Hellma GmbH) at RT. The samples were excited with a 375 nm EPL picosecond pulsed diode laser from Edinburgh Instruments Ltd.

Protein concentrations were determined with the Pierce BCA protein assay kit (Thermo Scientific/Dreieich, Germany). Pure recombinant DsrL proteins were quantified on the basis of their calculated extinction coefficients at 280 nm (67,600, 48,455, and 50,865 M^–1 cm^–1 for AvDsrL-1A CtDsrL-1B DaDsrL-2, respectively). UV-visible absorbance spectroscopy was carried out at 20°C on a Specord 210 UV/Vis spectrophotometer (Analytik Jena/Jena, Germany). The protein samples were prepared in 50 mM potassium phosphate buffer, pH 7.0, and assembled in a quartz glass cuvette (Hellma Analytics/Müllheim, Germany) in the Coy anaerobic chamber. The cuvette was sealed with air-tight septa and titanium(III) citrate (Zehnder and Wuhrmann, 1976 (link)) as reductant and potassium ferricyanide as oxidant were added via a gas-tight Hamilton syringe. All spectra were normalized to their absorption at 750 nm.

VIPP1 (Preps #4, #5, and #6) and liposomes were mixed at 4 °C with protein and lipid concentrations of 0.4 mg/mL and 0.15 mg/mL, respectively, and incubated for 20 min at 22 °C before being transferred to a 3 mm × 3 mm quartz glass cuvette (Hellma Analytics) for measurement. TRF anisotropy measurements were recorded at 22 °C on a FluoFit 300 fluorescence lifetime spectrometer (PicoQuant) using time-correlated single photon counting. The excitation source was a pulsed LED emitting at λ_exc = 281 nm ± 5.5 nm. Emission was detected at λ_em = 355 nm ± 5 nm. Fluorescence decays were recorded with horizontal and perpendicular orientations of polarizers in the excitation and emission paths, respectively, each for 5 min. Photons were counted for 100 ns after each pulse in 4000 channels of 25 ps. The measurements were repeated after the addition of Mg²⁺ to a final concentration of 10 mM and an incubation at 22 °C for 20 min. Fluorescence anisotropy decays were fitted by a bi-exponential decay curve with FluoFit (PicoQuant) using data from 3–40 ns after excitation. Two rotational correlation times were obtained, where the fast one, corresponding to segmental tryptophan motions, was essentially constant at values < 0.5 ns, while the slow one, reflecting larger-scale protein motions, was exploited to probe VIPP1 binding to liposomes.