Structural composition of betaI- and betaII-proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.     

Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication

Inverse circular dichroism (CD) spectra are presented for each of the five major secondary structures of proteins: alpha-helix, antiparallel and parallel beta-sheet, beta-turn, and other (random) structures. The fraction of the each secondary structure in a protein is predicted by forming the dot product of the corresponding inverse CD spectrum, expressed as a vector, with the CD spectrum of the protein digitized in the same way. We show how this method is based on the construction of the generalized inverse from the singular value decomposition of a set of CD spectra corresponding to proteins whose secondary structures are known from X-ray crystallography. These inverse spectra compute secondary structure directly from protein CD spectra without resorting to least-squares fitting and standard matrix inversion techniques. In addition, spectra corresponding to the individual secondary structures, analogous to the CD spectra of synthetic polypeptides, are generated from the five most significant CD eigenvectors.     

Circular dichroism spectra of glutamate dehydrogenase plus reduced nicotinamide adenine dinucleotide.

Circular dichroism (CD) spectra are reported for various concentrations of glutamate dehydrogenase in order to determine any role of protein aggregation on NADH-binding spectra. These CD spectra do appear to be sensitive to enzyme aggregation. These spectra raise some doubt about previous interpretation of CD spectra as direct evidence for a second NADH binding-site.     

Circular dichroism of nucleic acid monomers. I. Calculated adenosine and 2′-deoxyadenosine CD spectra

A combination of the DeVoe and Kirkwood polarizability concepts is developed to calculate CD spectra of nucleic acid monomers. The method is perfectly general and applies to any system where the constituents have absorption properties which are widely separated in terms of frequency. The theory is applied to calculate the CD spectra of adenosine and 2′-deoxyadenosine conformers. Bond polarizabilities are evaluated for the ribosyl moiety of adenosine, as a function of glycosidic rotational angles and polarizability anisotropies. It is found that a wide range of C-C and C-O bond polarizabilities give similar CD results. Isotropic atom polarizabilities are also evaluated. It is found that the CD results using these polarizabilities do not differ significantly from those obtained with bond polarizabilities. The CD spectra of adenosine and 2′-deoxyadenosine are calculated for three x-ray diffraction determined geometries: A-form RNA, B-form DNA, and C-form DNA. The results indicate that the monomer CD spectra are strongly dependent on the precise geometry and appear to be of importance in understanding the spectra of oligomers and polymers. The deoxyadenosine conformers are found to have calculated CD spectra which are less intense than those of the ribosyl conformers. These results indicate that the measured differences between the CD magnitudes of ribo- and deoxyriboadenosine are due to the presence or absence of the 2′-hydroxyl. Weighted averaged adenosine CD spectra are calculated with the aid of probability distributions from conformational energy calculations. The results suggest a new method for obtaining empirical monomer parameters for use in optical calculations. The calculations in this paper indicate for the first time that DeVoe theory, in combination with the Kirkwood theory, provides a useful method for the calculation of the CD spectra of nonpolymeric molecules.     

Circular dichroism spectroscopy of fluorescent proteins

Circular dichroism (CD) spectra have been obtained from several variants of green fluorescent protein: blue fluorescent protein (BFP), enhanced cyan fluorescent protein (CFP), enhanced green fluorescent protein (GFP), enhanced yellow fluorescent protein (YFP), all from Aequorea victoria, and the red fluorescent protein from the coral species Discosoma (DsRed). We demonstrate that CD spectra in the spectral fingerprint region of the chromophore yield spectra that after normalization are not coincident with the normalized absorbance spectra of GFP, YFP and DsRed. On the other hand, the CD spectra of BFP and CFP coincide with the absorbance spectra. The resolution of absorption and CD spectra into Gaussian bands confirmed the location of the different electronic band positions of GFP and YFP as reported in the literature using other techniques. In the case of BFP and CFP the location of Gaussian bands provided information of the vibrational progression of the electronic absorption bands. The CD spectrum of DsRed is anomalous in the sense that the major CD band has a clear excitonic character. Far-UV CD spectra of GFP confirmed the presence of the high beta-sheet content of the polypeptide chain in the three-dimensional structure.     

Extending CD spectra of proteins to 168 nm improves the analysis for secondary structures

The CD spectra for 10 proteins with known secondary structure have been extended from 178 to 168 nm. Combined with the data for 6 other proteins investigated previously, this produces a basis set of 16 proteins, which can be used to analyze CD spectra for secondary structure. Extending the spectra adds another CD band to the data and increases the information content from the equivalent of five to six. Analyzing the CD for each of the 16 proteins in the basis set with the 15 other proteins shows a modest improvement in the prediction of secondary structure with the extended CD spectra.     

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.     

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.     

Accuracy of protein secondary structure determination from circular dichroism spectra based on immunoglobulin examples

Strong contribution of the aromatic amino acid side chain chromophores to the far-UV circular dichroism (CD) spectra substantially distorts a relatively weak CD signal originating from beta sheet, the main type of immunoglobulin secondary structure. In this study we compared the secondary structure calculated from the far-UV CD spectra with the X-ray data for three antibody Fab fragments. Calculations were performed with three different algorithms, using two sets of reference proteins. Low standard deviations between all six estimates indicate stable mathematical solutions. Despite pronounced differences in the shape and amplitude of the CD spectra, we found a strong correlation between CD and X-ray data in the secondary structure for every protein studied. The number and average length of the secondary structure elements estimated from the CD spectra closely resemble those of the X-ray data. Agreement between spectroscopic and crystallographic results demonstrates that modern methods of secondary structure calculation are resilient to distortions of the far-UV CD spectra of immunoglobulins caused by aromatic side chain chromophores.     

Detection of conjugated C16 PUFAs in rat tissues as possible partial beta-oxidation products of naturally occurring conjugated linoleic acid and its metabolites

In a previous paper, we showed that naturally occurring conjugated linoleic acid (CLA) from butter fat is metabolized in vivo to higher metabolites such as conjugated diene (CD) 18:3, CD 20:3 and CD 20:4, all the while retaining the conjugated diene structure. In this paper, we describe the detection of two more metabolites with characteristic conjugated diene UV spectra. HPLC retention times, UV and MS spectra identified the CLA metabolites as CD 16:2 and CD 16:3. The accumulation of CD 16:2 was significantly higher than that of CD 16:3 in all tissues examined. Tissue distributions of CD 16:2 and CD 16:3 were similar, with plasma and adipose tissue showing the highest levels, while kidney had the lowest and the liver an intermediate level. CD 16 fatty acids accounted for about 20% of the total CLA metabolites. The kidney, however, was an exception where CD 16 fatty acids accounted for only 11% of total metabolites. Analyses of liver lipid classes showed that CD 16:2 and CD 16:3 were preferentially incorporated into neutral lipids. This preferential incorporation was very similar to CLA as shown previously. We hypothesize that CD 16:2 and CD 16:3 may be derived from partial beta-oxidation of CLA and CD 20:4, respectively, even though we cannot rule out that CD 16:3 may also be derived from CD 18:3 and CD 20:3. Incubation of skin human fibroblasts from X-linked adrenoleukodystrophy (ALD) patients with c9,t11 CLA showed that CD 16:2 formation in ALD cells was about 50% lower than control cells. This result may tempt to hypothesize that, at least in part, CD 16:2 is beta-oxidized in peroxisomes.     

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes.

Circular dichroism (CD) spectra were measured for cytochromes P-450 (P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in alpha-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45--50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250--310 nm) and weaker negative CD spectra associated with Soret transitions for PBP-448 ([theta] = 50 000) and MCP-448 ([theta] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.     

Bacteriochlorophyll organization and energy transfer kinetics in chlorosomes from Chloroflexus aurantiacus depend on the light regime during growth

We have used measurements of fluorescence and circular dichroism (CD) to compare chlorosome-membrane preparations derived from the green filamentous bacterium Chloroflexus aurantiacus grown in continuous culture at two different light-intensities. The cells grown under low light (6 mol m^–2 s^–1) had a higher ratio of bacteriochlorophyll (BChl) c to BChl a than cells grown at a tenfold higher light intensity; the high-light-grown cells had much more carotenoid per bacteriochlorophyll.The anisotropy of the Q_Y band of BChl c was calculated from steady-state fluorescence excitation and emission spectra with polarized light. The results showed that the BChl c in the chlorosomes derived from cells grown under high light has a higher structural order than BChl c in chlorosomes from low-light-grown cells. In the central part of the BChl c fluorescence emission band, the average angles between the transition dipole moments for BChl c molecules and the symmetry axis of the chlorosome rod element were estimated as 25° and 17° in chlorosomes obtained from the low- and high-light-grown cells, respectively.This difference in BChl organization was confirmed by the decay associated spectra of the two samples obtained using picosecond single-photon-counting experiments and global analysis of the fluorescence decays. The shortest decay component obtained, which probably represents energy-transfer from the chlorosome bacteriochlorophylls to the BChl a in the baseplate, was 15 ps in the chlorosomes from high-light-grown cell but only 7 ps in the preparation from low-light grown cells. The CD spectra of the two preparations were very different: chlorosomes from low-light-grown cells had a type II spectrum, while those from high-light-grown cells was of type I (Griebenow et al. (1991) Biochim Biophys Acta 1058: 194–202). The different shapes of the CD spectra confirm the existence of a qualitatively different organization of the BChl c in the two types of chlorosome.Abbreviations BChl bacteriochlorophyll - CD circular dichroism - DAS decay associated spectrum - PMSF phenylmethylsulfonyl fluoride     

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region. In spite of the close geometric similarity of the beta turns of these two groups of peptides, their CD spectra are quite distinct. Furthermore, comparison of our data with the CD spectra of published models for beta-turn structures suggests that it may not be possible to characterize the contribution of all beta turns to the CD spectra of proteins by a single model curve. the CD spectra of model beta turns will be more useful in characterizing the folding of oligopeptides and sequence polypeptides, where a single type of turn is present.     

Comparative circular dichroism studies of an anti-fluorescein monoclonal antibody (Mab 4-4-20) and its derivatives.

This study presents circular dichroism (CD) spectra of a high-affinity monoclonal anti-fluorescein antibody (Mab 4-4-20), its Fab fragments, and corresponding single-chain antibody (SCA). In the region 200-250 nm, the differences in the CD spectra between these proteins reflect the uneven distribution of chromophores (tryptophan and tyrosine) rather than a major conformational change. On the basis of near-UV CD spectra, binding of the hapten fluorescein to these protein antibodies elicits an increased asymmetry in the microenvironment of the chromophoric residues in contact with the hapten and also perturbs the interface between VL and VH domains. The hapten-binding site provides a chiral microenvironment for fluorescein that elicits a pronounced induced fluorescein CD spectrum in both the visible and UV regions. In contrast to the parent molecules, SCA is thermolabile. Our results demonstrate that (1) UV CD spectra are useful for assessing the chromophoric microenvironment in the binding portion of antibodies and (2) the extrinsic fluorescein hapten CD spectra provide information about the interaction of hapten with the binding pocket.     

Circular dichroism of rhodopsins and porphyropsins

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 321–700 nm: an α peak at about 520 nm and a small β peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the α peaks of the porphyropsins are larger than the α peaks of the rhodopsins, while the β peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blue-shifted from the corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250–320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.     

Nucleotides. VI. Syntheses and spectral properties of some deazaadenylyl-deazaadenosines (dinucleoside monophosphates with unusual CD-spectrum) and closely related dinucleoside monophosphates.

Nine dinucleoside phosphates containing 1-deaza-(1A) and 3-deazaadenosine (3A) were prepared. Hypochromicity and CD spectra of these dimers were determined. It was found that varying degrees of base-stacking are operative with these oligonucleotides and their CD spectra fall into three classes. The first class CD spectra which are more or less similar in profile to those of adenylyl-(3'-5')-adenosine includes the CD spectra of 1A2'p5'A, 1A3'p5A, 3A2'p5'A and 3A3'p5'A. The second class includes the CD spectra of A2'p5'1A and A3'p5'1A whose characteristic is that the positive Cotton band appears in the range of 280-310 nm. The third type CD spectra has the characteristics that the negative Cotton band appears in the longer wavelength region and th CD spectra are similar in profile to those of L-adenylyl-(3'-5')-L-adenosine which has the "left-handed helical" conformation. The CD spectra of A2'p5'3A, A3'p5'3A and 3A3'p5'A belong to this class. Another salient observation emerging from the CD-determination is that 3A3'p5'3A has the spectrum quite different from that of poly 3-deazaadenylic acid.     

Changes in structure and stability of calbindin-D(28K) upon calcium binding

Calbindin-D(28K) is a biologically important protein required for normal neural function and for the transport of calcium in epithelial cells of the intestine and kidney. We have used fluorescence and circular dichroism (CD) spectroscopy to characterize the effects of calcium binding on the structure and stability of calbindin. Ca(2+) titration monitored by fluorescence spectroscopy reveals the presence of two classes of calcium-binding sites with association constants approximately 10(7.5) and approximately 10(8.9)M(-1). CD spectra in the far-UV spectral range show minor changes upon Ca(2+) titration, implying that the secondary structure of calbindin-D(28K) is not greatly affected. On the basis of the CD spectra in the near-UV spectral range, we conclude that the tertiary structure is more sensitive to Ca(2+) addition. The most significant change occurs between pCa 7.0 and pCa 8.0. The variations in the protein thermostability are correlated with those in the near-UV CD spectra. The enthalpy changes upon heat denaturation of calbindin in the apo-state are characteristic of proteins containing several weakly interacting domains with similar thermodynamical properties. Thus, calcium binding by calbindin-D(28K) largely affects the local structure around the aromatic residues and the thermal stability of the protein; the changes in the secondary structure are insignificant.     

Circular dichroism of some mononucleosides.

The circular dichroism (CD) and absorption spectra of uridine, thymidine, purine ribonucleoside, and the four adenine derivatives 2′-deoxyadenosine, adenosine, adenosine-3′,5′-cyclic phosphate, and arabinosyl adenine were measured in water at pH 7 and pH 2. The absorption and CD spectra of the pyrimidines were simultaneously fitted to four Gaussian bands, and the dipole and rotational strengths of the electronic transitions determined. Adenine-derivative CD spectra were determined by computer averaging six runs. The spectra showed CD bands at 268, 226, 209, and 195 nm. The band at 226 nm probably is an n–π* transition; the band at 209 nm cannot be detected without a computer. The CD and absorption spectra of purine ribonucleoside indicate three transitions in the 230–310-nm region.     

Circular dichroism studies of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with DNA

The circular dichroism spectra of complexes of the antibiotics daunomycin, nogalamycin, chromomycin, and mithramycin with calf thymus DNA have been measured over a range of drug/DNA ratios. The similarity of the CD spectra of bound chromomycin and mithramycin suggests that they have very similar binding sites, which produce strong effects on the CD spectra of the bound drugs, and remove the differences arising from local stereochemistry in the free drugs. It was found that it was not possible to predict whether the antibiotics intercalated, from studies of the CD spectra alone, even when comparisons were made with the CD spectra of aminoacridine–DNA complexes with intercalating or nonintercalating ligands.     

Bioinformatics analyses of circular dichroism protein reference databases

MOTIVATION: Circular dichroism (CD) spectroscopy has become established as a key method for determining the secondary structure contents of proteins which has had a significant impact on molecular biology. Many excellent mathematical protocols have been developed for this purpose and their quality is above question. However, reference database sets of proteins, with CD spectra matched to secondary structure components derived from X-ray structures, provide the key resource for this task. These databases were created many years ago, before most CD spectrophotometers became standardized and before it was commonplace to validate X-ray structures prior to publication. The analyses presented here were undertaken to investigate the overall quality of these reference databases in light of their extensive usage in determining protein secondary structure content from CD spectra. RESULTS: The analyses show that there are a number of significant problems associated with the CD reference database sets in current use. There are disparities between CD spectra for the same protein collected by different groups. These include differences in magnitudes, peak positions or both. However, many current reference sets are now amalgamations of spectra from these groups, introducing inconsistencies that can lead to inaccuracies in the determination of secondary structure components from the CD spectra. A number of the X-ray structures used fall short on the validation criteria now employed as standard for structure determination. Many have substantial percentages of residues in the disallowed regions of the Ramachandran plot. Hence their calculated secondary structure components, used as a foundation for the reference databases, are likely to be in error. Additionally, the coverage of secondary structure space in the reference datasets is poorly correlated to the secondary structure components found in the Protein Data Bank. A conclusion is that a new reference CD database with cross-correlated, machine-independent CD spectra and validated X-ray structures that cover more secondary structure components, including diverse protein folds, is now needed. However, that reasonably accurate values for the secondary structure content of proteins can be determined from spectra is a testament to CD spectroscopy being a very powerful technique.