Fs5 fluorescence spectrometer

NMR spectra were recorded on a 400 MHz Bruker UltraShield spectrometer using MeOD as the solvent. Chemical shifts were reported in ppm and were referenced to tetramethylsilane (internal) for ¹H and 85% H₃PO₄ (external) for ³¹P-NMR. Unless mentioned otherwise, all the NMR spectra were measured at 298 K. The mass spectra were measured in the positive ion mode. A methanol solution of AuNCs (≈0.5 mg mL⁻¹) was introduced into an Agilent 6530 Accurate Mass Q-TOF LC-MS system (with Agilent 1260 HPLC) via flow injection. The ESI mobile phase was made of acetonitrile/water (50/50) with ≈0.05% formic acid at a flow rate of 200 μL min⁻¹. The gas temperature of the ESI source was 130 °C at a flow rate of 8 L min⁻¹. The fragmentor voltage was set at 50 V, skimmer at 65 V, and the capillary voltage (V_cap) at 3500 V. The mass range measured was up to 20 000 for MS. The assignments were based on high resolution m/z values and isotopic distributions. UV-Vis-NIR spectra were measured in methanol solution of [Au₁₃–X₂] on an Agilent Technologies Cary 5000 UV-Vis spectrophotometer. The solution PL and QY measurements were performed on an Edinburgh Instruments FS5 fluorescence spectrometer. [Au₁₃–X₂] clusters were dissolved in methanol for measurements. The PLQYs were measured by using an FS5 Spectrometer with a built-in integrating sphere.

The photoluminescence spectra and lifetimes of the solids were measured with an FLS-980 fluorescence spectrometer (Edinburgh instruments, Livingston, UK) at room temperature. The excitation and the emission slit widths were 2 nm. The photoluminescence spectra of the solutions were measured with an FS-5 fluorescence spectrometer (Edinburgh instruments) at room temperature. The excitation and the emission slit widths were 5 nm. The photoluminescence quantum yields were measured with a Quantaurus-QY absolute photoluminescence quantum yield spectrometer (Hamamatsu Photonics, Hamamatsu, Japan) at room temperature. Photographs of the solid powders under natural light and UV light were taken with a Mi 10 Ultra smartphone (Xiaomi, Beijing, China). Parameters for photos taken under natural light: f/1.85, 1/50 s, ISO 1198, and 6.78 mm (equivalent focal length 24 mm). The parameters for the photos taken under UV light: f/1.85, 1/9 s, ISO 339, and 6.78 mm (equivalent focal length 24 mm).

Fluorescence emission and excitation spectra of the samples were measured with an FS5 fluorescence spectrometer and FS980 fluorescence spectrometer (Edinburgh Instruments, Livingston, UK). The excitation and the emission slit widths were 3 nm. The lifetimes were measured with an FS980 fluorescence spectrometer (Edinburgh Instruments, UK) and the luminescence quantum yields were measured with an absolute quantum yield tester (Quantaurus-QY, Hamamtsu, Japan). The photographs of the solutions under UV light (365 nm) were taken with a camera (D600, Canon, Tokyo, Japan) in a dark room. The exposure time was 1/4 s, and the ISO was 1600 unless otherwise stated.

According to the predicted results, a series of mutants were constructed using alanine-scanning site-directed mutagenesis by replacing the predicted key amino acids (L19, F352, N382, M383, N494, Q558, E568). All the primers used in this study were listed in Supplementary Table 1. The recombinant pET28a⁽⁺⁾-welR plasmid was used as the template. E. coli DH5α and E. coli BL21(DE3) strains were used for plasmid construction and protein expression, respectively. The expression and purification of protein mutants were performed as previously mentioned.
The structures of WelR and mutant proteins were analyzed by UV and FL spectroscopy. The concentrations of all proteins were similar but different, ranging from 80 to 110 μg/mL. Specifically, the concentrations of WelR, L19A, F352A, N382A, M383A, N494A and E568A were 110.5, 108.8, 96.4, 82.3, 83.4, 87.7, and 100.6 μg/mL, respectively. The solutions were an elution buffer. UV-visible absorption spectra were measured on a UV-visible spectrophotometer (TU-1810, Beijing Purkinje General Instrument Co. Ltd., China). FL spectra were recorded on an FS5 fluorescence spectrometer (Edinburgh Instruments, England), and the wavelength of the excitation light was 282 nm.

Fluorescence measurements were performed at 25 °C using an FS5 fluorescence spectrometer (Edinburgh Instruments, Livingston, UK) equipped with a 1 cm path length quartz cell. The excitation wavelength was 280 nm, and the emission wavelength was recorded between 290 and 500 nm with excitation and emission slit widths set at 5 nm. The prepared samples were subjected to a 5-fold dilution using the acetic acid–sodium acetate buffer with the same pH to measure fluorescence [32 (link)].