Circular dichroism 44

Circular dichroism (CD) is the differential absorption between left and right circularly polarized light (AL and AR) as a function of the wavelength. CD can be

used for the measurement of chiral molecules or complexes as long as one of the interacting species is chiral, since the left and right circularly polarized light components are absorbed to different extents. All biological systems and many non-living ones are chiral. The main applications of CD spectroscopy in this project include determining conformational changes in proteins, or changes of protein folding as the result of ligand interactions as well as structural predictions when the crystal structures are not available. According to Beer-Lambert’s law, the differential absorption is given by:

whereΔε is the differential molar extinction coefficient, cis the concentration (mol dm–3) andlis the path length (cm).

Also, the recombined differentially absorbed circularly polarized light components produce radiation that is elliptically polarized (i.e., traces out an ellipse). A CD instrument reports the signal as either the difference in absorbance, ΔA, of the two components, ΔA=AL–AR, or as the ellipticity in degrees,θ(θ= tan–1(b/a), whereb

and a are the minor and major axes of the resultant ellipse). There is a simple numerical relationship between ΔA and θ (θ in degrees), i.e. θ = 32.98 ΔA. In this project, CD spectra were collected on a Jasco J-815 spectropolarimeter, and ellipticityθwere reported as results.

CD of proteins

Proteins are made up of amino acids which are intrinsically chiral (with the exception of glycine). Secondary structure motifs within the peptide sequence such as the α-helix and β-sheet impose additional chirality on the peptide sequence. At present the main use of CD in the study of proteins is as an empirical gauge of protein structure and conformation using the CD induced into the backbone peptide region from 190 nm to 240 nm. In the absence of any contributions to the CD from side chain transitions, this has proved to be a very successful approach. Distinctive CD spectra (figure 3.1.2.1) have been described for pure conformations such as the

α-helix and β-sheet, β-turns, and also the random coil. The CD spectrum of a native protein is then the sum of the appropriate percentages of each component spectrum.

There are a range of different computer programs available for determining the percentage of different structural motifs from the CD spectrum. Generally, the algorithms use a library of well known peptide sequence or proteins with

well-defined secondary structures, and then a deconvolution of the data allows us to estimate the proportions of different secondary structure.

Figure 3.1.2.1 Far-UV CD spectra associated with various types of secondary structure of

protein: solid curve,α-helix; long dashes, anti-parallelβ-sheet; dots, type Iβ-turn, dots and short dashes, random coil (Johnson 1990).

α-helix: The α-helix is the dominant secondary structure in many proteins and on average accounts for about one-third of the residues in globular proteins. Theα-helix is a well defined motif, and the CD spectrum is characterised by negative peaks at 222 nm and 208 nm with separate maxima of similar magnitude (corresponding to a n π*transition and part of the first ππ* transition respectively) and a positive peak at 190 nm which corresponds to the remainder of the first π  π* transition.

The helical length determines the intensity of the positive peak at 190 nm. The 208 nm peak is unique to theα-helix.

β-sheet: The CD spectra of β-sheets are not as well defined as α-helix due to the practical reason that they are less soluble in solvents with a good UV transmission and due to the intrinsic reason that β-sheets can be parallel or antiparallel and of varying lengths and widths. However, the common characteristics of CD spectra of β-sheet may shown to be a negative band at approximately 216 nm and a positive band near 195 nm.

β-turn:The labelβ-turn usually is used to include all possible turns in a protein, not simply the ones that enable a single strand to become an antiparallel β-sheet. Because of this range of structures, the CD of β-turns is not really well defined. A typicalβ-turn CD spectrum has a weak red-shifted negative band near 225 nm due to n  π* transitions, a strong positive band between 200 nm and 205 nm corresponding to π  π* transitions, and a strong negative band between 180 nm and 190 nm.

Random coil: When we refer to random coils, we refer to the parts of a folded protein that do not fit into any of the categories previously discussed, so not really random. The CD spectrum of a random coil protein is characterised by a strong negative band below 200 nm, a positive band at about 218 nm and sometimes a very weak negative band at 235 nm.