Circular Dichroism Spectroscopy

Open nitrogen flow valve and turn on CD spectrometer. Open Spectrum Manager software. Allow nitrogen to flow through the optics for 10 min.

Select spectrum measurement option. Make sure that all precheck parameters display “OK” before proceeding with the instrument.

Add 200 μL of solvent to the 0.1 mm quartz cuvette (see Note 18). Use the sample holder to verify the volume is higher than the light source opening. Place cuvette into the sample chamber with the sample block. Return the sample chamber cap. Select experimental parameters for a CD scan, including: wavelength range (260 nm—start, 190 nm—end); response (4 s); accumulation (2 scans/experiment). Solvent spectrum should display a line with no or very little slope.

Remove solvent from cuvette and add 200 μL of the protein sample. Run CD scan.

Subtract baseline solvent spectrum from protein sample. Smooth curve for final output.

Convert data to mean residue ellipticity, θ_MRE, using a spreadsheet program. Run SELCON3 or other program for deconvolution of CD data. Output will include global secondary structure and random coil content of the protein.

CD spectroscopy is a useful technique in determining the secondary structure of a protein. Common features of α-helix (minima at 222 nm and 208 nm; maximum at 190 nm), β-sheet, (minimum at 218 nm; maximum at 196 nm), and random coil (minimum at 195 nm; maximum at 212 nm) can be seen from the CD spectra. The spectra can be deconvoluted to determine the global secondary structure content of a protein, using various methods and software (recently reviewed by N.J. Greenfield [106]). The assumption used by all methods is that the observed spectrum is a linear combination of the spectra of its secondary structural components plus a noise contribution from aromatic groups and prosthetic groups. For example, a CD measurement that has strong minima at 222 nm and 208 nm is expected to have a large fraction of α-helix structure. This is helpful when verifying the fold type and stability of a protein sample. These analyses are implemented by software such as SELCON3 [107] which takes mean residue ellipticity, or θ_MRE, as input. SELCON3 will analyze these data and report the content of each secondary structure in the analyte.

The combination of bioinformatic approaches, protein function prediction and computational docking has allowed the identification of new members of the macrodomain family based on protein sequence alone. These sequences have also been used to develop homology models. To validate these predictions, a system of biochemical and biophysical approaches for characterizing protein structure and ligand interactions may be employed. These methods are useful for the study of proteins with no previously known structural or functional data, as described here for a putative macrodomain in the bat coronavirus HKU4.