Cary eclipse fluorimeter

The synthesis and characterisation of 1 were reported previously.3 All measurements were performed in aqueous buffer solution (pH 7.0, I=0.05 m, sodium cacodylate/HCl buffer). The UV/Vis spectra were recorded on a Varian Cary 100 Bio spectrometer and fluorescence spectra were recorded on a Varian Cary Eclipse fluorimeter in quartz cuvettes (1 cm). Under the experimental conditions used, the absorbance of 1 was proportional to its concentration.
Polynucleotides were purchased as noted: poly dGdC—poly dGdC, poly dAdT—poly dAdT, poly A—poly U, poly A, poly G, poly C, poly U (Sigma), calf thymus (ct)‐DNA (Aldrich) and dissolved in sodium cacodylate buffer, I=0.05 m, pH 7.0. The ct‐DNA was additionally sonicated and filtered through a 0.45 mm filter to obtain mostly short (approx. 100 base pairs) rod‐like B‐helical DNA fragments.54 The polynucleotide concentration was determined spectroscopically55 as the concentration of phosphates (corresponds to c(nucleobase)).
Bovine Serum Albumin (BSA) (Sigma–Aldrich) was dissolved in sodium cacodylate buffer, I=0.05 m, pH 7.0 and its concentration determined spectroscopically using a NanoDrop spectrophotometer at 280 nm using its molar extinction coefficient of 43 824 m⁻¹ cm⁻¹.

Total HK activity was performed by the reduction of NAD⁺ measured fluorimetrically. Fluorescence was detected at an excitation wavelength of 352 nm (slit 10 nm) and an emission wavelength of 464 nm in the coupled reaction with exogenous G6PDH from L. mesenteroides, as described previously [39 (link)]. The measurement was done using a Varian Cary Eclipse fluorimeter. To assess HK activity, the cells (around 3 × 10⁶ cells) were permeabilized in the presence of 2 mM pyruvate, 2 mM malate and 10 mM glutamate with 0.002% digitonin. Then, the cells were centrifuged at 4°C, resuspended in the assay medium and kept on ice. The assay medium contained 10 mM Tris-HCl pH 7.4, 320 mM mannitol, 24 mM MgCl₂, 0.08 mM EDTA, 1 mM EGTA, 8 mM Pi, 1 mM ATP, 10 µM AP5A, 1 U/ml G6PDH, 1 mM β-NAD+ and the reaction started upon the addition of 5 mM glucose.

Cells isolated from Pocillopora damicornis (mixed cells: coral host and symbiotic algae) suspended in sterile artificial seawater were analyzed using a Varian Cary Eclipse Fluorimeter with the following settings: scan from λ_ex 200–700/λ_em 350–900, excitation slit 20 nm, emission slit 5 nm, scan rate 9600.00 nm/min, data interval 2.00 nm, average time 0.0125 s, auto excitation filter, open emission filter, medium PMT voltage, no corrected spectra. The resulting data was used to produce the excitation–emission matrix presented in Fig. 3.

A Cu(I) stock solution
(typically 50 mM) prepared using [Cu(CH₃CN)₄]PF₆ (Merck) in 100% anhydrous acetonitrile was diluted
into 20 mM HEPES pH 7.5 plus 200 mM NaCl.^{13 (link),14 (link),38 (link),51 (link)} The Cu(I)
concentration of this working stock was determined with BCS that forms
the [Cu(BCS)₂]^3– complex with an ε
value of 12 500 M^–1 cm^–1 at 483 nm.^{38 (link),52 (link)} This was routinely compared to
the total Cu concentration quantified using AAS. To monitor the effect
of Cu(I) binding to proteins, the buffered Cu(I) solution was titrated
into apo-Csp3s (typically ∼5 μM) and the formation of
S(Cys)→Cu(I) ligand-to-metal charge-transfer bands was observed
by UV/vis spectroscopy. Fluorescence was also measured during Cu(I)
titrations on a Cary Eclipse fluorimeter (Varian), exciting at 280
nm and monitoring the emission in the 400–700 nm range with
excitation and emission slits set to 10 and 20 nm, respectively.^{13 (link),51 (link)} Cu(I) was also added to protein (∼2.5 μM) plus ∼100
μM BCA, which forms the [Cu(BCA)₂]^3– complex with an ε value of 7700 M^–1 cm^–1 at 562 nm.^{32 (link),40 (link),52 (link)} This experiment was performed as either a titration for RkCsp3 [equilibration for each Cu(I) addition is complete
in ∼15 min] or by setting up a series of mixtures that were
incubated and measured for up to 48 h for SlCsp3
(slower equilibration).

Using the DPH probe, the anisotropy values of cell membranes of RBCs modified with the test compounds were measured. RBCs for this analysis were prepared according to the method described in subsection 2.5.3. Then, the DPH probe was added to RBCs with a haematocrit of 0.2%. The final concentration of the probe in the sample was 1.3 μM. The mixture was incubated protected from light at 37°C for 30 min. Then, the compounds dissolved in ethanol were added. The compounds were tested at 20, 60, and 100 μM concentrations. Samples with compounds and control samples with alcohol of the appropriate concentration were incubated at 37°C for 1 h. Measurements in triplicate were made in quartz cuvettes using a CARY Eclipse fluorimeter (Varian, San Diego, CA, United States) at 37°C. The excitation wavelength for the DPH probe is λ_exc = 360 nm and the emission wavelength is λ_em = 426 nm. Based on the changes in DPH on the intensity under polarized light, the anisotropy value was determined according to the formula used in our previous publication (Pruchnik et al., 2018 (link)).

Light scattering measurements used a Varian Cary Eclipse fluorimeter at 30 °C, with all samples in a buffer of 50 mM Tris–HCl, pH 7.3, 20 mM KCl, 3 mM magnesium acetate. Samples were pre-incubated at 30 °C, centrifuged in a microfuge and the supernatant transferred to a quartz microcuvette. GTP was added to a final concentration of 1 mM and light scattering recorded for at least 30 min. The GTP-induced change in light scattering in the presence of EzrA and FtsZ was normalized relative to the average GTP-induced scattering change for FtsZ alone.