Determining the CD Spectrum of a Protein

Plane-polarized radiation comprises two circularly polarized vectors of equal intensity, one right-handed and the other left-handed (Fig. B3.5.3A), which are separately measured in the CD spectrometer by means of a photoelastic modulator. A chromophore situated 

In both the far- and near-UV regions, CD spectra can be used empirically as fingerprints of a particular protein, with the spectrum resulting from the aromatic residues being rather more specific and hence diagnostic. The far-UV spectra, however, can provide information about the protein conformation in terms of its secondary structure. As for fluorescence spectroscopy or any spectroscopic method, the sample needs to be chemically pure and homogeneous. 

The output of CD spectrometers is in two alternative but related types of unit. The difference in absorbance with respect to left- and right-handed circularly polarized 

Ellipticity is usually used for far-UV measurements. Because essentially every residue in a protein is associated with a peptide bond, spectra in this region can be directly compared for different proteins by calculating the mean residue ellipticity, 

For example, if the ellipticity at 209 nm using a 1-mm path-length cell for a solution containing 0.361 mg/ml protein is -61.7 mdeg, and the mean residue molecular weight of the protein is 110, then [0] = -18.8 x 103 deg cm2/dmol. 

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Signals in the near UV region (250 00 nm) of the CD spectrum of a protein predominantly arise from absorption of the aromatic side chains of Trp, Tyr, and Phe. The dichroism in this region is highly dependent on the tertiary structure of the protein. For this reason, the near UV CD spectrum is often used to monitor changes in the native environment of proteins that result from instability. For example, Lam, Patapoff, and Nguyen used the near UV CD spectrum of IFN-y to determine that there was an incompatibility of the protein with a combination of excipients (succinate, benzyl alcohol) that resulted in a loss of structure. 

The analysis of CD spectra to obtain information about protein secondary structure has been reviewed extensively. It is convenient to represent the CD spectrum of a protein as a column vector, c, where the elements of the matrix are the values of the CD spectrum at a set of wavelengths, generally spaced at equal intervals, for example, at every nanometer over a range from 180 to 260 nm. Similarly, we can represent the secondary structure of a protein by a column matrix, f, with m rows, each element of which is the fraction of residues in the protein in one of the secondary structural elements under consideration. The problem of determining the protein secondary structure can then be formulated as a matrix equation ... 

Circular dichroism measurement is a convenient method to detect the conformation of molecules,especially protein molecules [1,2]. We used this method to pigment-protein complexes,PSII particles and chloroplasts of higher plants. The helix, yfl-pleated sheet, -twist,and unordered coil forms of proteins each has a characteristic circular dichroism (CD) spectrum, the CD spectrum of a protein molecule is the sum of contributions of these forms. We can determine the fractions of each form in a protein molecule from the CD spectrum of the protein. 

Figure 5-12. CD spectra of native and unfolded proteins. The CD spectra of proteins in the far UV is determined by the absorption bands of the peptide bond. The environment of the peptide bond in a region of defined secondary structure is significantly different from that in a disordered structure. Hence, the CD spectrum of a...

For proteins, it is the amide chromophore and the long-range order or lack of it that is responsible for the characteristic spectra of each of the secondary structural elements. A number of approaches have been employed to analyze the ORD or CD spectrum of a protein for the type and content of component secondary structures utilizing a set of basis spectra. The basis spectra are derived from the ORD/CD spectra of proteins from which the secondary structural content has been determined by X-ray crystallography. It is the solution of the linear combination of the basis spectra that gives the relative proportion for each of the secondary structural elements in the protein. 

Of the visible spectroscopic techniques, CD spectroscopy has seen the most rapid and dramatic growth. The far-UV circular dichroism spectrum of a protein is a direct reflection of its secondary structure [71]. An asymmetrical molecule, such as a protein macromolecule, exhibits circular dichroism because it absorbs circularly polarized light of one rotation differently from circularly polarized light of the other rotation. Therefore, the technique is useful in determining changes in secondary structure as a function of stability, thermal treatment, or freeze-thaw. 

The helix contents of five peptide fragments from the protein thermolysin have been determined by CD and NMR in both water and 30% TFE. 85 The helix content was obtained from CD by the method of Chen et a I.162 and the NMR method utilized chemical shifts. 84 Four of the five peptides correspond to helical regions in the intact protein, and one corresponds to an Q-loop. 86 The rms difference between CD and NMR helix contents for the five peptides under the two conditions is 7.5%. One peptide shows the largest deviations (0 vs 13% in water, 45 vs 62% in 30% TFE). If it is excluded, the rms deviation decreases to 4%. The peptide showing the largest deviation, residues 258-276 from the thermolysin sequence, has two Tyr and one Phe (with the Phe adjacent to one of the Tyr in the sequence), and therefore it has an above-average content of aromatic amino acids, which can perturb both the CD spectrum and NMR chemical shifts. Of the other four peptides, three have a single aromatic residue and the fourth has two aromatics. 

A similar secondary structure is predicted for the 11S globulin. However, differences appear in the tertiary structure as shown by the CD spectrum in the 240-320 nm region. Both tyrosine and tryptophan appear to be buried in the hydrophobic interior of the 11S globulin. CD spectra for both proteins in 0.1 N sodium hydroxide also reveal significant differences in tertiary structure (11). Tyrosine residues in the 7S protein appear to ionize much more readily than in the 11S protein as determined by the magnitude of the positive peak at 253 nm. 

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