Thermal unfolding of WT and ionic lock mutant GRK5 was monitored by recording variations in ellipticity at 222 nm as a function of temperature in 1.0°C increments from 20 to 70°C using a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control system. Measurements were conducted using a 0.1 cm quartz cuvette (Starna Cells, Inc.) with GRK5 at a final concentration of 5 μM in phosphate-buffered saline solution (3.2 mM Na2HPO4, 0.5 mM KH2PO4, pH 7.4, 135 mM NaCl). The cooperativity index, n, describing the sigmoidicity of the thermal denaturation curve was calculated by applying the Hill equation, n = (log 81)/log(T0.9/T0.1), where T0.1 and T0.9 are the temperatures where the fractional completions of the unfolding transition are 0.1 and 0.9, respectively.
0.1 cm quartz cuvette
Manufactured by Starna Cells
The 0.1 cm quartz cuvette is a laboratory equipment designed for spectroscopic analysis. It is made of high-quality quartz material and has a pathlength of 0.1 centimeters. This cuvette is used to hold liquid samples during various spectroscopic measurements, such as UV-Vis, fluorescence, or absorption spectroscopy.
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3 protocols using 0.1 cm quartz cuvette
1
Thermal Unfolding of WT and Mutant GRK5
2
Circular Dichroism Analysis of Portal Proteins
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CD scans were acquired on a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control system using a 0.1 cm quartz cuvette (Starna Cells, Inc.)70 (link). Assays were carried out using purified portal-602 and portal-725 dissolved at 1 μM final concentration in 10 mM HEPES, pH 7.4 and 70 mM NaCl. CD scans were measured between 196 and 260 nm at 5 °C. Secondary structure content estimated from the measured ellipticity using the K2d method35 (link).
3
Circular Dichroism Spectroscopy of His-UOX
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CD spectra were obtained using a Jasco J-815 spectropolarimeter. His-UOX was diluted to 10 μM in UOX reaction buffer and dialyzed extensively in 10 mM sodium phosphate pH 8.0. His-UOX (1 μM final) was mixed with Xe/O2-sparged phosphate buffer, sparged directly with Xe/O2 as described above, or unsparged, and 1400 μL of this mixture was loaded into a 0.1 cm quartz cuvette (Starna Cells, Inc.) and immediately sealed. Temperature in the cell was maintained at 22.0°C by a Jasco PFD-425S/15 Peltier device. Eight scans from 260 to 190 nm were performed at 20 nm/min with a 0.5 nm step size. Each condition above was measured three independent times including buffer-only baseline scans. Scans were accumulated, averaged, and subtracted from the average baseline.
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