Circular dichroism and magnetic circular dichroism spectra of chlorophylls a and b in nematic liquid crystals. II. Magnetic circular dichroism spectra

Absorption and magnetic circular dichroism (MCD) spectra are reported for chlorophyll (Chl) a and Chl b dissolved in nematic liquid crystal solvents. The spectra were measured with the dye molecules oriented uniaxially along the direction of. the magnetic field and measuring light beam. It is significant that under such conditions the MCD spectra recorded in the wavelength region of the Q and Soret bands of the chlorophyll are essentially unchanged with respect to rotation of the sample cell around this axis, even though there is almost complete orientation of the chlorophyll molecules by the liquid crystals. The MCD spectra of Chl a and b in the nematic liquid crystal solvents used in this study are surprisingly similar to the spectra obtained under isotropic conditions. These results illustrate an important technique with which to examine the optical spectra of dyes oriented in liquid crystal matrices in which the anisotropic effects can be reduced the negligible proportions by the application of a strong magnetic field parallel to the direction of the measuring light beam. The first deconvolution calculations are reported that describe the deconvolution of pairs of absorption and MCD spectra, in the Q and B band regions, for both Chl a and b. The spectral analysis to obtain quantitative estimates of transition energies was accomplished by carrying out detailed deconvolution calculations in which the both the absorption and MCD spectral envelopes were fitted with the same number of components; each pair of components had the same hand centres and bandwidth values. This procedure resulted in an assignment of each of the main transitions in the absorption spectra of both Chl a and b. Chl a is clearly monomeric, with Qy, Qx, By and Bx located at 671, 582, 439 and 431 nm, respectively. Analysis of the spectral data for Chl b located Qy, By and Bx, at 662, 476 and 464 nm, respectively.     

Protein secondary structure from circular dichroism spectra

Circular dichroism spectra of proteins are extremely sensitive to secondary structure. Nevertheless, circular dichroism spectra should not be analyzed for protein secondary structure unless they are measured to at least 184 nm. Even if all the various types ofβ-turns are lumped together, there are at least 5 different types of secondary structure in a protein (α-helix, antiparallelβ-sheet, parallelβ-sheet,β-turn, and other structures not included in the first 4 categories). It is not possible to solve for these 5 parameters unless there are 5 equations. Singular value decomposition can be used to show that circular dichroism spectra of proteins measured to 200 nm contain only 2 pieces of information, while spectra measured to 190 nm contain about 4. Adding the constraint that the sum of secondary structures must equal 1 provides another piece of information, but even with this constraint, spectra measured to 190 nm simply do not analyze well for the 5 unknowns in secondary structure. Spectra measured to 184 nm do contain 5 pieces of information and we have used such spectra successfully to analyze a variety of proteins for their component secondary structures.     

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.     

Membrane-phorbol ester interactions monitored by circular dichroism

The interaction of phorbol 12,13-dibutyrate (PDBu), 12-O-retinoylphorbol 13-acetate (RPA) and 12-O-tetradecanoylphorbol 13-acetate (TPA) with L-alpha-phosphatidylserine-containing small unilamellar vesicles or erythrocyte ghosts was monitored by circular dichroism (CD). No change in the CD spectra of PDBu was observed upon binding, while RPA and TPA spectra were slowly affected by the interaction. The changes in RPA and TPA spectra were assigned to the embedding of these molecules in the membrane bilayers. In the presence of 10(8) cells/ml, after one minute incubation, about 2 to 5% of the amount of phorbol ester added is embedded in the membrane. It is suggested that either phorbol esters entering the membrane is not a prerequisite for protein kinase C activation or the amount of phorbol esters necessary to activate protein kinase C is very small.     

Analyses of circular dichroism spectra of membrane proteins

Circular dichroism (CD) spectroscopy is a valuable technique for the determination of protein secondary structures. Many linear and nonlinear algorithms have been developed for the empirical analysis of CD data, using reference databases derived from proteins of known structures. To date, the reference databases used by the various algorithms have all been derived from the spectra of soluble proteins. When applied to the analysis of soluble protein spectra, these methods generally produce calculated secondary structures that correspond well with crystallographic structures. In this study, however, it was shown that when applied to membrane protein spectra, the resulting calculations produce considerably poorer results. One source of this discrepancy may be the altered spectral peak positions (wavelength shifts) of membrane proteins due to the different dielectric of the membrane environment relative to that of water. These results have important consequences for studies that seek to use the existing soluble protein reference databases for the analyses of membrane proteins.     

On the analysis of membrane protein circular dichroism spectra

Analysis of circular dichroism spectra of proteins provides information about protein secondary structure. Analytical methods developed for such an analysis use structures and spectra of a set of reference proteins. The reference protein sets currently in use include soluble proteins with a wide range of secondary structures, and perform quite well in analyzing CD spectra of soluble proteins. The utility of soluble protein reference sets in analyzing membrane protein CD spectra, however, has been questioned in a recent study that found current reference protein sets to be inadequate for analyzing membrane proteins. We have examined the performance of reference protein sets available in the CDPro software package for analyzing CD spectra of 13 membrane proteins with available crystal structures. Our results indicate that the reference protein sets currently available for CD analysis perform reasonably well in analyzing membrane protein CD spectra, with performance indices comparable to those for soluble proteins. Soluble + membrane protein reference sets, which were constructed by combining membrane proteins with soluble protein reference sets, gave improved performance in both soluble and membrane protein CD analysis.     

Using circular dichroism spectra to estimate protein secondary structure

Circular dichroism (CD) is an excellent tool for rapid determination of the secondary structure and folding properties of proteins that have been obtained using recombinant techniques or purified from tissues. The most widely used applications of protein CD are to determine whether an expressed, purified protein is folded, or if a mutation affects its conformation or stability. In addition, it can be used to study protein interactions. This protocol details the basic steps of obtaining and interpreting CD data, and methods for analyzing spectra to estimate the secondary structural composition of proteins. CD has the advantage that measurements may be made on multiple samples containing < or =20 microg of proteins in physiological buffers in a few hours. However, it does not give the residue-specific information that can be obtained by x-ray crystallography or NMR.     

Chirality of aromatic bis-imides from their circular dichroism spectra

It is demonstrated that chirality of molecules composed of 1,2,4, 5-benzenetetracarboxydiimide (pyromellitic diimide) or 1,4,5, 8-naphthalenetetracarboxydiimide units is reflected by their exciton Cotton effects. The analysis is based on the calculated (ZINDO/S) excited states of the model diimide chromophores 1a and 2a. Rotation of the diimide chromophores around the C-N bond in diimides 3-5 is evaluated from the dynamic (1)H NMR data. A comparison of chiroptical properties of bis-diimides 3-5 with the CD spectra of bis-imides 6-8 is also presented. Copyright 2000 Wiley-Liss, Inc.     

Vibrational circular dichroism spectra of unblocked proline oligomers.

Vibrational Circular Dichroism (VCD) spectra of unblocked L-proline oligopeptides, (Pro)n n = 3 to 7, dissolved in D2O are reported. For these oligomers, the VCD spectra can be attributed to a conformational dominance of the trans amide conformation with subunits interrelated by a left-handed twist, particularly for the longer oligomers. As a function of oligomer length, formation of this conformation starts at n = 3; and by n = 5 a spectrum closely resembling that of the poly-L-proline II helix in shape and magnitude is seen. The VCD data are compared with previous (Pro)n results using IR, CD, Raman and NMR spectroscopies, and reasons for the variations in interpretation are discussed.     

Determination of protein secondary structure from circular dichroism spectra. III. Protein-derived base spectra of circular dichroism for antiparallel and parallel beta-structures

Protein-derived basic CD spectra for alpha-helix, antiparallel and parallel beta-structures, beta-bends and irregular form of proteins have been determined from the experimental CD spectra of six (myoglobin, lysozyme, ribonuclease A, papain, lactate dehydrogenase, subtilisin BPN') or seven (glyceraldehyde-3-phosphate dehydrogenase added) reference proteins and the analysis of the X-ray data. The secondary structures of thirteen proteins (seven reference and six additional ones) have been analysed using the basic CD spectra thus obtained. The data obtained have been compared with the results of the X-ray data analysis. It is shown that the accuracy of determination of the beta-structure and beta-bends contents using our basic CD spectra is about 2-3 times better than using the basic spectra reported by Chang et al. (Analyt. Biochem. 91, 13-31, 1978).     

Absorption flattening in the circular dichroism spectra of small membrane fragments

The inhomogeneous distribution of chromophore occurring in a particulate suspension can result in a reduction in the apparent molar ellipticity recorded in circular dichroism (CD) spectra. The possibility of such a systematic error has often been a matter of concern when CD spectra of cell membrane proteins are recorded. The recent publication of CD spectra for bacteriorhodopsin in native and sonicated membranes, in detergent-solubilized form, and reconstituted into small unilamellar vesicles [Mao, D., & Wallace, B. A. (1984) Biochemistry 23, 2667-2673] gives a unique opportunity to apply the theoretical analysis of Gordon and Holzwarth [Gordon, D. J., & Holzwarth, G. (1971) Arch. Biochem. Biophys. 142, 481-488] so as to provide a definitive answer to the question of whether absorption flattening is significant for membrane particles. We show here that the data of Mao and Wallace can be combined with the theoretical analysis of Gordon and Holzwarth to rule out significant absorption flattening effects over the range 200-240 nm for submicrometer-sized membranes. In addition, the results show that absorption flattening can be disregarded even at 190 nm for membranous material in the size range below 100 nm. The demonstration that there are no major flattening effects in the CD spectra of bacteriorhodopsin, particularly in the region of 200-240 nm, means that the experimental spectra are incompatible with the proposal that this transmembrane protein contains seven transmembrane helices.     

Computer-aided analysis of infrared, circular dichroism and absorption spectra

Marquardt and Powell optimization methods without constraintson the optimized spectral parameters were employed for decompositionof complex i.r., c.d. and absorption spectra into componentbands. The procedure resolved experimental spectra into eightcomponent bands and it can be easily adjusted for a larger setof component bands. The CPU time required for achievement ofsatisfactory convergence of parameters for eight component bandsis rather large even when using mainframe computers and thereforedivision of spectra into a few non-overlapped parts is advisable.The program also can be used for calculation of absorption,c.d. and difference spectra from formatted raw spectral data. Received on January 13, 1986; accepted on April 7, 1986     

Detection of vibron phenylalanine bands in circular dichroism spectra for collagen

For the first time the individual positive circular dichroism peaks at 268 nm, 262 nm and 256 nm have been found for collagen. These peaks are due to the vibronic nature of a weak pi-pi* phenylic chromophore transition of phenylalanine and the unique structural organization of collagen where water molecules are an essential element of the triple helix.     

Wavelength shifts in solid-state circular dichroism spectra: a possible explanation

We have devised an artificial sampling approach generating "absorption flattening" (AF) on the UV-Vis spectrum of a solution of a chiral compound: Tris (ethylendiamine)cobalt(III) chloride (Lambda-Co(en)(3)Cl(3)). We have observed a concomitant red shift of the CD maxima. Related CD and absorption spectra have been calculated from spectra recorded on diluted homogeneous solutions, thus monitoring the effect of AF on both types of data. Experimental data are in good agreement with calculated spectra. Simulations with suitable bandshapes show that the red shift of the CD spectrum is due to AF. On the basis of these results, we conclude that AF is an important cause of distortions in CD spectra for inhomogeneous samples. Plans to compensate or at least to take into account this effect are presented.     

Investigation of huge negative circular dichroism spectra of some nucleoproteins

Under certain conditions of preparation, DNA, whether free or complexed with polylysine or histone KAP (I, fl), produce huge negative circular dichroism (CD) spectra with maxima at about 270nm. In order to investigate the cause of these spectra, reconstituted polylysine-DNA complex was used as a model system. It was found that the CD change of DNA in the complex is not a linear function of the fraction of base pairs bound. Such a CD spectrum is not changed despite dilution up to 128 folds for as long as 12 hours. Difference CD spectra taken between free DNA and any of the complexes are qualitatively the same, and are similar to those of free DNA and nucleohistone KAP (Fasman et al., Biochemistry 9, 2814-2822, 1970), free DNA and direct mixed polylysine-DNA complexes, or free DNA in high salt (Chang et al., Biochemistry12, 3028-3032, 1973). The suggestion is made that this CD spectrum might be caused by specific conformational changes in DNA, perhaps belonging to the family of B to C transitions followed by a further structural distortion of DNA due to aggregation of the nucleoprotein molecules.     

Analyzing protein circular dichroism spectra for accurate secondary structures.

We have developed an algorithm to analyze the circular dichroism of proteins for secondary structure. Its hallmark is tremendous flexibility in creating the basis set, and it also combines the ideas of many previous workers. We also present a new basis set containing the CD spectra of 22 proteins with secondary structures from high quality X-ray diffraction data. High flexibility is obtained by doing the analysis with a variable selection basis set of only eight proteins. Many variable selection basis sets fail to give a good analysis, but good analyses can be selected without any a priori knowledge by using the following criteria: (1) the sum of secondary structures should be close to 1.0, (2) no fraction of secondary structure should be less than -0.03, (3) the reconstructed CD spectrum should fit the original CD spectrum with only a small error, and (4) the fraction of alpha-helix should be similar to that obtained using all the proteins in the basis set. This algorithm gives a root mean square error for the predicted secondary structure for the proteins in the basis set of 3.3% for alpha-helix, 2.6% for 3(10)-helix, 4.2% for beta-strand, 4.2% for beta-turn, 2.7% for poly(L-proline) II type 3(1)-helix, and 5.1% for other structures when compared with the X-ray structure.     

Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin

Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1L(a) and the 1L(b) electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1L(b) spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1L(b) component from the rest of the 1L(b) was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1L(a) and 1L(b) components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1L(b) component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra.