Samples were prepared dissolving the lyophilized protein in 7 M GdmCl to eliminate any possible non-specific aggregate induced by the freeze-dry procedure, and then dialyzed extensively against 20 mM sodium borate at pH 8.5. For chemical denaturation experiments, protein samples at 50 μM were prepared in solutions of GdmCl at different concentrations in 20 mM sodium borate buffer at pH 8.5. The experiments were performed at 298 K, and the unfolding reaction was monitored by tryptophan fluorescence. The chemical denaturation experiments (Fig. 2g in main article) were fitted to a two-state unfolding transition that rendered the following parameters for wild-type CI2: Cm = 4.07 M and ΔG0 = 30.9 kJ mol−1; and for CI2eng: Cm = 1.28 M and ΔG0 = 10.9 kJ mol−1. For thermal denaturation experiments, protein samples at different concentrations were prepared in 20 mM sodium borate buffer at pH 8.5. Fluorescence experiments were performed collecting the total fluorescence emission after excitation at 280 nm in a Jobin Yvon Fluorolog-3 spectrofluorometer from Horiba. Far-UV CD experiments were performed measuring the molar ellipticity of the sample from 190 to 250 nm every 1 nm on a Chirascan CD spectrometer from Applied Photophysics.
Jobin yvon fluorolog 3 spectrofluorometer
Manufactured by Horiba
The Jobin Yvon Fluorolog-3 is a spectrofluorometer designed for fluorescence measurements. It is capable of steady-state fluorescence, time-resolved fluorescence, and phosphorescence measurements. The instrument features high sensitivity and a wide range of excitation and emission wavelengths.
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5 protocols using jobin yvon fluorolog 3 spectrofluorometer
1
Protein Unfolding Dynamics Characterization
2
Photoluminescence of CNA-Encapsulated Dye
3
Fluorescence Lifetime of HSA-MY Interaction
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The fluorescence lifetime of HSA combined with different concentrations of MY was measured by Jobin Yvon Fluorolog-3 spectrofluorometer (Horiba, LesUlis, FRA), and the proportions were 1 : 0, 1 : 1.5, 1 : 3, respectively.
4
Rap1 Binding Dynamics on Nucleosomes
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All measurements were performed using a Fluorolog®-3 Horiba Jobin Yvon spectrofluorometer, in T50 buffer (10 mM Tris pH 8, 50 mM NaCl) 60 μl total volume. Nucleosomes (final concentration of 25-30 nM) and Rap1 (0, 1, 2, 5, 10 equivalents) were mixed by pipetting in T50 buffer and left for 10 min room temperature to bind. Fluorescence emission spectra are taken from 585 nm to 700 nm (1 nm increments) using 578 nm as excitation wavelength. Spectra for DNA only, T50 only and donor only samples were taken. For a given sample, NaCl was added to 800 mM to observe nucleosome disassembly. FRET efficiency was calculated from donor emission: with FDA denoting donor emission in the presence of acceptor, and FD denoting donor emission in the donor-only sample. Additionally, reactions were loaded onto 0.5x TBE 5% polyacrylamide gels to check binding.
5
Steady-State Fluorescence Spectroscopy of PrxF31W
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Concentrated PrxF31W was dialyzed in 1 L of buffer (25 mM Tris pH 7.0) for 1.5 h to ensure adequate buffer exchange. The protein was then diluted to the appropriate stock concentration. Steady‐state fluorescence spectra were measured on a Fluorolog‐3 Horiba Jobin Yvon spectrofluorometer (Edison, NJ) using a 10 × 3.3 mm quartz cuvette to hold the sample. The samples were excited at 280 nm, the excitation and emission slits were set to a 2 nm bandpass. All equilibrium folding and unfolding experiments in this work was performed upon protein samples with a final concentration of 5 μM PrxF31W in the buffer of choice. All samples were equilibrated at room temperature (20°C) for 1 h prior to the scan. In the unfolding experiments, Urea stock solutions were prepared for each predetermined concentration of potassium fluoride. The final urea concentrations were determined by refractive index (Grimsley et al., 2006 (link)) using 115 V AC/DC Refractometer purchased from Fisher Scientific (Free Lawn, NJ) to verify the final concentration of denaturant post data acquisition. The data were analyzed with Sigma Plot (Point Richmond, CA) software.
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