Stocks of POPC and PET1 were prepared in chloroform and 1 mM sodium phosphate (pH 7.4) buffer, respectively. An aliquot of POPC was dried under a stream of argon gas before placed in a desiccator for at least 2 h. The POPC film was resuspended with 1 ml of 1 mM NaPi pH 7.4 buffer and extruded through a 100 nm Nuclepore Track-Etch Membrane (Whatman) to produce LUVs. PET1 was diluted to a working concentration of 7 μM peptide suspended in 20 mM sodium phosphate pH 7.5 or 20 mM sodium acetate pH 4.3. PET1 was incubated with LUVs at a 150:1 lipid to peptide molar ratio. Samples were recorded on a Jasco J-815 CD spectrometer using a 2 mm quartz cuvette (Starna Cells Inc). All conditions were averaged over two technical replicates. Appropriate buffer backgrounds were collected on the same day and subtracted appropriately.
2 mm quartz cuvette
Manufactured by Starna Cells
Sourced in United States
The 2 mm quartz cuvette is a laboratory instrument used for spectroscopic analysis. It is made of quartz material and has a path length of 2 millimeters. The cuvette is designed to hold liquid samples for UV-Vis or other types of spectroscopic measurements.
Automatically generated - may contain errors
3 protocols using 2 mm quartz cuvette
1
POPC Liposome Preparation and PET1 Binding
2
Steady-State UV-Vis Spectrophotometry
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Steady-state spectra
were collected using either a Cary 60 or 5000 UV–Vis spectrophotometer
(Agilent Technologies, Santa Clara, CA). Spectra were typically collected
from 200 to 800 nm in 1 nm steps. Solutions were contained in a 2
mm quartz cuvette (Starna Cells, Atascadero, CA) and had a maximum
absorbance of ca. 0.3 in the range of 500–700 nm.
were collected using either a Cary 60 or 5000 UV–Vis spectrophotometer
(Agilent Technologies, Santa Clara, CA). Spectra were typically collected
from 200 to 800 nm in 1 nm steps. Solutions were contained in a 2
mm quartz cuvette (Starna Cells, Atascadero, CA) and had a maximum
absorbance of ca. 0.3 in the range of 500–700 nm.
3
CD Analysis of LASV FD Mutants
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CD measurements
were performed on a Jasco J-810 spectrophotometer (Jasco; Easton,
MD, USA) with a 2 mm quartz cuvette (Starna Cells; Atascadero, CA,
USA). Samples contained 0.8 mM POPC:POPG (65:35) with 20 μM
LASV FD (WT) or each mutant. All experiments were carried out at 23
°C in 1 mM HMA, 10 mM NaCl, and pH 4 unless indicated otherwise.
For the pH titration, 100 mM HCl was added stepwise until the desired
pH was achieved, which was confirmed with a calibrated pH probe. Spectra
were collected from 260 to 198 nm with a step size of 1 at 20 nm/min
and averaged over three accumulations. Measurements were taken for
the buffer alone containing 0.8 mM SUVs and subtracted from each sample,
then smoothed using the program CDToolX.42 (link) Spectra were converted to mean residue ellipticity (MRE) units and
the helical percentage was calculated from the MRE at 222 nm via the
following equation: fH = , where θC = 2,220–53T,
θH = (250T – 44,000)*(1 – ), T is the temperature in Celsius, and
n is the number of residues in the protein.28 (link),43 (link),44 (link)
were performed on a Jasco J-810 spectrophotometer (Jasco; Easton,
MD, USA) with a 2 mm quartz cuvette (Starna Cells; Atascadero, CA,
USA). Samples contained 0.8 mM POPC:POPG (65:35) with 20 μM
LASV FD (WT) or each mutant. All experiments were carried out at 23
°C in 1 mM HMA, 10 mM NaCl, and pH 4 unless indicated otherwise.
For the pH titration, 100 mM HCl was added stepwise until the desired
pH was achieved, which was confirmed with a calibrated pH probe. Spectra
were collected from 260 to 198 nm with a step size of 1 at 20 nm/min
and averaged over three accumulations. Measurements were taken for
the buffer alone containing 0.8 mM SUVs and subtracted from each sample,
then smoothed using the program CDToolX.42 (link) Spectra were converted to mean residue ellipticity (MRE) units and
the helical percentage was calculated from the MRE at 222 nm via the
following equation: fH = , where θC = 2,220–53T,
θH = (250T – 44,000)*(1 – ), T is the temperature in Celsius, and
n is the number of residues in the protein.28 (link),43 (link),44 (link)
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