CD measurements were performed on an Applied Photo Physics Chirascan CD spectropolarimeter with a 0.1 cm path length quartz cuvette in buffers as specified in the Protein expression and purification section. Data were collected every 1 nm with a scan rate of 120 nm/min; for each measurement two scans were recorded. Data presented are averaged from at least two separate measurements of different protein preparations. Thermal measurements were performed in a temperature range from 10 to 85 °C with a 0.7 °C/min heating rate, data acquisition every 1 °C. The mean residue molar ellipticity (MRE) of proteins was calculated as described35 (link). Here we use the units of deg × cm2 × dmol-1, rather than the units deg × cm² × dmol-1 res-1. The helical content of proteins was calculated from values of the amide n → π* transition at 222 nm ([MRE222]), as previously described35 (link).
Chirascan cd
Manufactured by Applied Photophysics
Sourced in United Kingdom
The Chirascan CD is a circular dichroism (CD) spectrometer manufactured by Applied Photophysics. It is used to measure the differential absorption of left and right circularly polarized light by chiral molecules. The Chirascan CD provides accurate and reliable measurements of the secondary structure of proteins and other biomolecules.
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12 protocols using chirascan cd
1
Circular Dichroism Spectroscopy Protocol
2
Far-UV CD Spectroscopy of PV Structures
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Far-UV CD spectra (190 to 260 nm) of PV monomers, dimers, and fibrils were collected at room temperature (21 ± 1 °C) using a Chirascan CD spectropolarimeter (Applied Photophysics) and a quartz cuvette with a path length of 1 mm. Five scans were collected and averaged for each sample at a time-per-point of 0.8 s, with a bandwidth of 1 nm and a step size of 1 nm. The signal of the buffer was subtracted, and the resulting intensities in millidegrees of the PV monomer and dimer samples were converted to mean residue molar ellipticity (degrees M−1 m−1).
3
Circular Dichroism Spectroscopy of Proteins
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CD measurements were performed on an Applied Photo Physics Chirascan CD spectropolarimeter with a 0.1-cm-path length quartz cuvette in 0.1 m NaCl, 10 mm sodium phosphate, pH 7.4 buffer. Data were collected every 1 nm with 30-s averaging time, each measurement being an average of two repeated scans. Data presented are averaged from at least two separate measurements of different protein preparations. Thermal measurements were performed in a temperature range from 10 to 85 °C with a 0.7 °C/min heating rate with data acquisition every 1 °C and 20-s averaging time. The sample cooling rate prior to measurement of refolded protein was ∼ 2 °C/min. The mean residue molar ellipticity of proteins was calculated as described (25 (link)). The helical content of proteins was calculated from values of the amide nπ* transition at 222 nm ([MRE222]) as described previously (25 (link)). Protein concentration was measured by absorption at 280 nm. Absorption coefficients were obtained from ProtParam software. Standard concentrations were in the range of 10–20 μm . In the salt dependence experiments, stock buffer (5 m NaCl, 10 mm sodium phosphate, pH 7.4) was mixed with stock protein solution to obtain desired salt and protein (10 μm ) concentration.
4
Far-UV Circular Dichroism Spectroscopy
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Far-UV circular
dichroism (CD) spectra
were recorded on a Chirascan CD spectrophotometer (Applied Photophysics,
Leatherhead, Surrey, UK) using a 0.1 mm path length cuvette. Spectra
shown are the average of three scans that were acquired over the range
of 200–260 nm with a bandwidth of 1 nm and a scan speed of
20 nm min–1. The buffer contribution was subtracted
from each sample. For the amphipol containing samples, buffer containing
the appropriate amount of A8-35 was used as the reference.
dichroism (CD) spectra
were recorded on a Chirascan CD spectrophotometer (Applied Photophysics,
Leatherhead, Surrey, UK) using a 0.1 mm path length cuvette. Spectra
shown are the average of three scans that were acquired over the range
of 200–260 nm with a bandwidth of 1 nm and a scan speed of
20 nm min–1. The buffer contribution was subtracted
from each sample. For the amphipol containing samples, buffer containing
the appropriate amount of A8-35 was used as the reference.
5
Far-UV CD Spectroscopy of Proteins
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Far-UV CD spectra were recorded on a Chirascan CD spectrophotometer (Applied Photophysics, Leatherhead, Surrey, UK) using a 1-mm path length cuvette. Proteins were buffer-exchanged into 10 mm sodium phosphate buffer, pH 7.0 and measured at a protein concentration of 10 μm . Three scans were acquired over the range 190–260 nm with a bandwidth of 2.5 nm and a scan speed of 1 nm/s. The three datasets were averaged, and the buffer contribution was subtracted to produce the final spectrum. Secondary structure content was estimated by uploading the data into DichroWeb (57 (link)) and using the CONTIN (58 (link)) algorithm.
6
Circular Dichroism Analysis of AAC(6')-Ii
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CD measurements were obtained using a ChiraScan CD instrument (Applied Photophysics, Leatherhead, Surrey, UK). Each sample contained 500 μL of protein solution consisting of AAC(6′)‐Ii at 0.5 mg·mL−1 and 100 μL of deionized water or 100 μL of 50 μm AcCoA. For measurements at different pH values, the protein solution was prepared in a 5 mm sodium‐phosphate buffer at pH 6 and subsequently mixed at a 1 : 1 ratio with a 50 mm sodium‐phosphate buffer at the desired final pH. The wavelength spectrum used extended from 180 to 260 nm in 0.5 nm steps. For temperature‐dependence studies, AAC(6′)‐Ii at 100 μg·mL−1 in sodium‐phosphate buffer at pH 6 in the absence or presence of 20 μm AcCoA was used. Changes in molar ellipticity (θ) were measured at wavelengths 191, 195, 208, 217, and 222 nm, while temperature was increased from 6 °C to 94 °C and then reduced to 6 °C in 2 °C per 2 min intervals.
7
Circular Dichroism Analysis of Proteins
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Circular dichroism measurements were performed on a Chirascan CD (Applied Photophysics) in 100 mM NaPi, 150 mM NaF pH 8.0. Data were collected at 0.5 nm intervals in the wavelength range of 180─260 nm at 20 °C, using a temperature-controlled chamber. 30 μL of 100 μM ACP5a, ACP5a E6761A/L6764N and VirD were analysed in a 0.01 cm cuvette, while 100 μL of 100 μM VirD E128Q were analysed in a 0.1 cm cuvette. Each spectrum (Supplementary Fig. 1 ) represents the average of three scans, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone.
8
Thermal Stability of HSP18 in NaCl
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The structural stability of HSP18 in the absence or presence of 0.5 M NaCl was determined using thermal denaturation experiments as described previously [29 (link)]. Briefly, HSP18 (0.2 mg/ml in 10 mM phosphate buffer, pH 7.5) was heated from 25–80°C with a ramp rate of 0.5°C/min and the alteration in ellipticity at 222 nm was recorded using Chirascan CD spectropolarimeter (Applied Photophysics, Leatherhead, UK). Mid-point transition or melting temperature (Tm) was estimated using sigmoidal analysis as described previously [29 (link)] and van’t Hoff equation was used to calculate the enthalpy change (ΔHvH) associated with the thermal transition of HSP18 in the absence and presence of 0.5 M NaCl.
9
Circular Dichroism Spectroscopy of Proteins
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CD was carried out on a Chirascan CD (Applied Photophysics). Data were collected at 0.5 nm intervals in the wavelength range of 200−260 nm at 20 °C, using a temperature-controlled chamber. A 0.01 cm cuvette containing 30 μL of protein sample at 50 μM was used for all the measurements. Each spectrum represents the average of three scans, and sample spectra were corrected for buffer background by subtracting the average spectrum of buffer alone.
10
Biophysical Characterization of Purified Proteins
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Purified protein (0.1 mg mL -1 ) in 10 mM potassium phosphate buffer pH 7.6 was used for CD measurements using the Chirascan CD spectrophotometer (Applied Photophysics). The secondary structure content was calculated from the far UV CD measurements using the CDNN software. The melting temperature (T m ) of the sample was determined using the Global3 software by comparing the spectra from 195 to 260 nm obtained by heating the sample at a rate of either 1 or 2 °C per min from 22 to 94 °C. In addition, the EcTFE and EcTFE-β were preincubated with varying concentrations of ATP or ADP on ice for 30 min and the CD measurements of these samples were recorded in a similar way.
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