Circular dichroism difference spectra

KD Philipson, VL Sato and K Sauer (1972) Exciton interaction in photosystem I reaction center from spinach chloroplasts. Absorption and circular dichroism difference spectra. Biochemistry 11 4591-4595... 

The UV spectrum of a complex conjugated molecule is usually observed to consist of a few broad band systems, often with fine structure, which may be sharpened up in non-polar solvents. Such a spectrum can often be shown to be more complex than it superficially appears, by investigation of the magnetic circular dichroism (MCD) spectrum, or by introduction of dissymmetry and running the optical rotatory dispersion (ORD) or circular dichroism (CD) spectrum. These techniques will frequently separate and distinguish overlapping bands of different symmetry properties <71PMH(3)397). 

The fluorescence spectrum of LHC la at 77 K showed a major emission band near 680 nm plus a weaker emission band near 730 nm. In contrast, the emission of the LHC Ib complex appeared centered almost entirely at 730 nm, as shown in Fig. 2 (B). As with the absorption spectrum, the fluorescence spectrum of LHC I is intermediate between those of the two subcomplexes. The circular-dichroism (CD) spectrum of LHC I (not shown) is drastically different from that of CC I, showing prominent optical activity at 648 and 485 nm, reflecting the presence of a significant amount of Chi b in the complex. As seen in Fig. 2 (C), the CD spectra ofLHC la and LHC Ib are similar to each other but differ in intensity, which probably reflects differences in pigment-pigment or pigment-protein interactions in them. The strong 340-nm band observed in LHC I fractions is absent in CC I which lacks Chi b. 

Figure 3 Top Effect of increasing Cd(II) binding, i.e., addition of 1-7 equivalents, on the circular dichroism (CD) spectrum of apoMT from rabbit liver at pH 8.4. Bottom Difference CD spectrum of CdyMT versus apoMT. The units are based on the protein concentration. Adapted from [43] with permission from the American Chemical Society copyright 1987.

Circular dichroism (c.d.) spectroscopy measures the difference in absorption between left- and right-circularly polarized light by an asymmetric molecule. The spectrum results from the interaction between neighboring groups, and is thus extremely sensitive to the conformation of a molecule. Because the method may be applied to molecules in solution, it has become popular for monitoring the structure of biological molecules as a function of solvent conditions. 

Figure 15. Circular dichroism of the C=0 C li peak (BE = 292.7 eV) in fenchone at three different photon energies, indicated, (a) Photoelectron spectrum of the carbonyl peak of the (1S,4R) enantiomer, recorded with right (solid line) and left (broken line) circularly polarized radiation at the magic angle, 54.7° to the beam direction, (b) The circular dichroism signal for fenchone for (1R,4A)-fenchone (x) and the (lS,41 )-fenchone (+) plotted as the raw difference / p — /rep of the 54.7° spectra, for example, as in the row above, (c) The asymmetry factor, F, obtained by normalizing the raw difference. In the lower rows, error bars are included, but are often comparable to size of plotting symbol (l/ ,4S)-fenchone (x), (lS,4R)-fenchone (+). Data are taken from Ref. [38],...

The Ca -ATPase has been crystallized in both conformations [119,152-155]. The two crystal forms are quite different [10,88-93,156-161], suggesting significant differences between the interactions of Ca -ATPase in the Ei and E2 conformations. Since the Ei-E2-transition does not involve changes in the circular dichroism spectrum of the Ca -ATPase [162], the structural differences between the two states presumably arise by hinge-like or sliding motions of domains rather than by a rearrangement of the secondary structure of 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 u.v.-visible spectrum of the 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl-methyl-cobinamide is very similar to methyl-cobin-amide itself and as a result this technique cannot be used to rigorously identify the spin labeled derivative. The spin labeled compound does show a spectral change with pH between pH 7.0 and pH 2.0 which methyl-cobinamide does not exhibit. Despite the similarities between methyl-cobinamide and nitroxylmethylcobinamide, the circular dichroism spectrum of the two derivatives are quite different. Fig. 23 shows the marked difference in C. D. spectra of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, methylcobinamide, and a methylcobinamide solution containing an equimolar amount of uncoordinated nitroxide. 

To obtain statistically significant comparisons of ordered and disordered sequences, much larger datasets were needed. To this end, disordered regions of proteins or wholly disordered proteins were identified by literature searches to find examples with structural characterizations that employed one or more of the following methods (1) X-ray crystallography, where absence of coordinates indicates a region of disorder (2) nuclear magnetic resonance (NMR), where several different features of the NMR spectra have been used to identify disorder and (3) circular dichroism (CD) spectroscopy, where whole-protein disorder is identified by a random coil-type CD spectrum. 

Because configurational information can be derived from optical rotatory dispersion and circular dichroism scans, considerable work has been conducted using these techniques to study the tetracyclines (32). The absolute configuration of CTC was determined using optical rotatory dispersion data (33) Spectral curves are presented in Figures 8 and 9. The circular dichroism spectrum is similar to that presented by Mitscher et al. (34), except that the values differ by a factor of about 1.5. In Table 3, data obtained by Mitscher and in FDA laboratories are compared. 

In a more recent study using circular dichroism, Pflumm and Beychok (313) have fitted the observed curve for RNase-A (see Figs. 11c and d) weighted mixtures of the characteristic bands for helix from poly-L-glutamic acid and / structure from poly-L-lysine. The data are compatible with 11.5% helix and 33% / conformation. Ribonuclease-S and RNase-A have almost identical CD spectra from 198 to 300 nm. The spectrum of S-protein is markedly different from the other two. 

M(A-A)3 complexes are optically active, and the problem can be remedied if a circular dichroism spectrum of one enantiomer can be measured and the sharp electronic lines identified. The bite and twist angles have very different effects on the rotational strengths. The twist angle has a marked effect that is quite plausible when one considers that a 60° twist causes conversion to the opposite enantiomer. Sign changes in the rotational strength can also occur at twist angles near / = 0°. 

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