1. Sample preparation

  a. Buffer composition

• Choose a buffer whose components have low absorption in the far-UV region. For a detailed list of commonly used buffer components and their absorption see (Micsonai, Bulyáki, and Kardos 2021). Phosphate buffer with the minimum amount of salt required for the protein is strongly preferred. For buffers with substantial absorbance in the far-UV use a cuvette with shorter pathlengths but be aware that this also requires higher protein concentrations.

• Avoid compounds with high absorption in the 180-260 nm range such as denaturing agents (GdnHCl and urea), reducing agents (dithiothreitol and mercaptoethanol) and organic solvents dimethyl sulfoxide (DMSO) or dimethylformamide (DMF). If necessary, use the denaturant dodine instead of GdnHcl or urea ( much lower concentrations of dodine are sufficient) and replace DTT and mercaptoethanol with Tris (2-carboxyethyl) phosphine (TCEP).

• Transfer the sample into the buffer to be used for the CD measurements either by dialysis or by using a centrifugal filter device. Dissolving lyophilized powder or diluting the protein from another buffer in the measurement buffer is not recommended as it can lead to contamination or a buffer composition differing from the reference buffer.

  b. Purity

• Make sure that the protein sample is homogenous and free of contaminants, such as other proteins and chiral biomolecules (e.g. nucleotides).

• Check the purity of your sample by SDS-PAGE, reversed phase HPLC, mass spectrometry, absorption spectroscopy, or other complementary methods.

• Unless the aim is to study protein aggregates or amyloid fibrils, remove any precipitated or aggregated proteins by centrifuging at >10,000 x g. If needed, use ultracentrifugation at 100,000 x g to remove protein oligomers.

• When studying protein aggregation or amyloid fibrils, homogenize samples thoroughly by pipetting or using ultrasonication and apply only a short centrifugation at low force to remove large aggregates.

  c. Protein tags

• Remove any tags or extensions from the protein of interest as these can affect the spectrum.

• If it is not possible to remove the tags, take their contribution into account when analyzing the data (number of residues, molecular weight, presumed contribution to the estimated secondary structure contents, possible effect on the secondary structure of the protein).

  d. Protein concentration

• It is essential to determine the protein concentration of the sample accurately!

• For proteins containing tryptophan and/or several tyrosine residues, concentration can be determined based on absorbance measured at 280 nm. The extinction coefficient can be obtained by using the ProtParam tool (https://web.expasy.org/protparam/).

• If the protein lacks tryptophan residues and contains no or very few tyrosine side chains, absorbance measured at 205 (Anthis and Clore 2013) or 214 nm (Kuipers and Gruppen 2007) should be used to calculate the protein concentration. The corresponding extinction coefficients can be retrieved from the BeStSel homepage (https://bestsel.elte.hu/extcoeff.php). The magnitude of the extinction coefficients at 205 and 214 nm makes it possible to determine the protein concentration directly on the CD sample.

2. Cuvettes/Cells

• Quartz cells can be used for recording spectra above 180 nm. For low sample volumes or measurements that go below 180 nm use calcium fluoride cells.

• Select the pathlength of the cell so that the product of the pathlength in mm and the protein concentration in mg/ml is around 0.1.

• If the measurement buffer has substantial absorbance in the far UV region, choose a cuvette with shorter pathlength and increase the protein concentration according to the relationship provided above.

• To record spectra down to 180 nm on conventional instruments, use cells with short pathlengths (10-50 μm) and adjust the protein concentration accordingly.

• Make sure that the cell is clean and lint-free. Use a proper detergent and lint-free lens cleaning wipes to clean the cells before each measurement.

3. Instrument status

• The instrument should be calibrated regularly according to the manufacturer’s instructions.

• Let the UV lamp warm up for an hour after turning it on. Start measuring only after the lamp has warmed up.

4. Measurement/instrumental parameters

• Collect data on the widest usable wavelength range from 260 nm down to at least 200 nm, but favorably to 190 or 180 nm. To measure CD data down to 175 nm use a synchrotron radiation CD (SRCD) instrument.

• Set the bandwidth to 1 nm or at most 2 nm.

• In continuous scanning mode choose a response or data integration time so that the wavelength is not shifted more than the value of the bandwidth during that time. (E.g. set response time to 0.5 sec when applying a scanning rate of 100 nm/min and 1 nm bandwidth.)

• Record and average several scans of each sample to achieve a better signal to noise ratio.

• Always record the baseline spectrum of the buffer using the very same settings as for the protein sample!

• Measure the CD spectrum of the buffer first. Estimate the usable wavelength range from the HT (high tension) values obtained, which should not exceed 50-60% of the maximum value provided by the manufacturer. Discard data measured at HT values above this limit.

5. Data
  a. Baseline subtraction

• Always correct the spectrum of the protein by subtracting the CD spectrum of the corresponding buffer (baseline)!

  b. Filtering

• Apply only moderate smoothing (Savitzky-Golay filtering) on the spectrum while taking care not to change any sharp component or steep part of the spectrum.

  c. Normalization

• The CD spectrum of the protein sample should be normalized for protein concentration, pathlength of the cell and the number of peptide bonds in the protein.

• If you use BeStSel for data analysis, you may upload the baseline-subtracted raw data and provide the required data on concentration, number of residues and pathlength and BeStSel will produce the normalized data for you (in Δε).

• Otherwise, normalize the baseline-corrected spectrum of the sample to obtain mean residue molar ellipticity [Θ]_MRE in units of deg·cm²·dmol^-1 according to the following relationship: [Θ]_MRE = Θ/(10·c·Nr·l), where Θ is the measured ellipticity in mdeg, c stands for the molar concentration of the protein, Nr is the number of residues in the protein, and l is the pathlength in cm.

• If you prefer the use of Δε (M^-1·cm^-1), Δε=[Θ]_MRE / 3298

References:

Anthis, Nicholas J., and G. Marius Clore. 2013. “Sequence-Specific Determination of Protein and Peptide Concentrations by Absorbance at 205 Nm.” Protein Science: A Publication of the Protein Society 22 (6): 851–58. https://doi.org/10.1002/pro.2253.

Kuipers, Bas J. H., and Harry Gruppen. 2007. “Prediction of Molar Extinction Coefficients of Proteins and Peptides Using UV Absorption of the Constituent Amino Acids at 214 Nm to Enable Quantitative Reverse Phase High-Performance Liquid Chromatography-Mass Spectrometry Analysis.” Journal of Agricultural and Food Chemistry 55 (14): 5445–51. https://doi.org/10.1021/jf070337l.

Micsonai, András, Éva Bulyáki, and József Kardos. 2021. “BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy.” Methods in Molecular Biology (Clifton, N.J.) 2199: 175–89. https://doi.org/10.1007/978-1-0716-0892-0_11.