Convex constraint analysis: a natural deconvolution of circular dichroism curves of proteins

A new algorithm, called convex constraint analysis, has been developed to deduce the chiral contribution of the common secondary structures directly from experimental CD curves of a large number of proteins. The analysis is based on CD data reported by Yang, J.T., Wu, C.-S.C. and Martinez, H.M. [Methods Enzymol., 130, 208-269 (1986)]. Application of the decomposition algorithm for simulated protein data sets resulted in component spectra [B (lambda, i)] identical to the originals and weights [C (i, k)] with excellent Pearson correlation coefficients (R) [Chang, C.T., Wu, C.-S.C. and Yang, J.T. (1978) Anal. Biochem., 91, 12-31]. Test runs were performed on sets of simulated protein spectra created by the Monte Carlo technique using poly-L-lysine-based pure component spectra. The significant correlational coefficients (R greater than 0.9) demonstrated the high power of the algorithm. The algorithm, applied to globular protein data, independent of X-ray data, revealed that the CD spectrum of a given protein is composed of at least four independent sources of chirality. Three of the computed component curves show remarkable resemblance to the CD spectra of known protein secondary structures. This approach yields a significant improvement in secondary structural evaluations when compared with previous methods, as compared with X-ray data, and yields a realistic set of pure component spectra. The new method is a useful tool not only in analyzing CD spectra of globular proteins but also has the potential for the analysis of integral membrane proteins.     

Calculated optical properties of 64 trinucleoside diphosphates

The optical rotatory dispersion and ultraviolet absorption spectra of the 64 trinucleoside diphosphates containing the bases A, U, C, and G have been calculated by using a simple semiempirical approach. These calculations accurately predict the optical properties of the nine trimers for which extensive experimental results are available. The computed optical data should be useful in the identification of oligonucleotides obtained in the course of sequence determination of ribonucleic acids and should simplify the determination of the concentration of oligonucleotides in aqueous solution. Additional calculations indicate that it should be possible to analyze most, mixtures of sequence isomers of trinucleoside diphosphates by direct, measurement of the ORD of the mixture at neutral pH.     

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.     

An infrared and circular dichroism combined approach to the analysis of protein secondary structure.

Selected regions of infarred (ir) and circular dichroism (CD) spectral data from 10 proteins were combined and analyzed by a factor analysis method. The regions consisted of the area normalized amide I region from 1700 to 1600 cm-1 for the ir spectra and from 178 to 240 nm for the CD spectra. Each CD spectrum was scaled by a factor of 0.5 before appending the data to the ir spectral data. The scaling factor was deemed necessary to account for relative intensity differences between the ir and CD data and provided nearly optimum agreement between secondary structure estimated by the combined approach to secondary structure determined by X-ray crystallography. The ir/CD combined approach to estimation of helix, beta-sheet, beta-turn, and other or undefined secondary structure agreed with X-ray crystallographic determined structure better than estimation using data from either method alone. Correlation coefficients between X-ray and ir/CD combined secondary structure determinations were 0.99 for helix, 0.90 for beta-sheet, 0.70 for beta-turn, and 0.78 for other structure. The four most significant eigenvectors or basis spectra from eigenanalysis of the ir/CD data are presented as well as generalized inverse spectra for four secondary structures.     

Polynucleotides. XXXI1. Synthesis of AUG analogs containing 8,2''-S-cycloadenosine, 8,5''-S-cycloadenosine, 8-bromoadenosin, 8-oxyadenosine and formycin in the first position of the codon.

Five AUG analogs having 8,2'-S-cycloadenosine (I), 8,5'-S-cycloadenosine (II), 8-bromoadenosine (III), 8-oxyadenosine (IV) and formycin (V) in the first position of ApUpG W were synthesized. 3'-Phosphates of I, II and V were synthesized by phosphorylation using cyanoethylphosphate and DCC. In the case of II, 2', 3'-cyclic phosphate was directly obtained. 3'-Phosphates, thus obtained, were properly protected on the 2'-OH and/or the N6-amino group and condensed with U(OBz)pGiBu(iBu)2 using DCC to give ApUpG analogs. Some properties on paper chromatography and electrophoresis, and the UV and CD spectra of these trinucleoside diphosphates are reported.     

Tchebichef image moment approach to the prediction of protein secondary structures based on circular dichroism

Circular dichroism (CD) spectroscopy is a widely used technique for the evaluation of protein secondary structures that has a significant impact for the understanding of molecular biology. However, the quantitative analysis of protein secondary structures based on CD spectra is still a hard work due to the serious overlap of the spectra corresponding to different structural motifs. Here, Tchebichef image moment (TM) approach is introduced for the first time, which can effectively extract the chemical features in CD spectra for the quantitative analysis of protein secondary structures. The proposed approach was applied to analyze reference set and the obtained results were evaluated by the strict statistical parameters such as correlation coefficient, cross‐validation correlation coefficient and root mean squared error. Compared with several specialized prediction methods, TM approach provided satisfactory results, especially for turns and unordered structures. Our study indicates that TM approach can be regarded as a feasible tool for the analysis of the secondary structures of proteins based on CD spectra. An available TMs package is provided and can be used directly for secondary structures prediction.     

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).     

Negative ion fast atom bombardment-tandem mass spectrometry for structural analysis of isoprenoid diphosphates

Applicability of negative ion fast atom bombardment (FAB)-tandem mass spectrometry (MS/MS) was examined in trace mixture analyses and structural assignments of some isoprenoid diphosphates. Negative ion FAB-MS spectra using a glycerol matrix of these isoprenoid diphosphates showed predominantly molecular ions (M-H)- together with fragment ions at m/z 177 (H3P2O7)-, 176 (H2P2O7)-, 159 (HP2O6)-, and 79 (PO3)- which were characteristic of the diphosphate ester moiety. The molecular ions did not overlap with peaks arising from any impurities even when crude sample such as butanol extracts from enzymatic reaction mixtures were directly analyzed without any purification. Moreover, collisionally activated dissociation spectra of the molecular ion showed many structurally significant fragment ions which enabled us to elucidate the structures of such irregular alkyl chain moieties as those having a homoisoprenoid skeleton or substituted structures. These studies indicate that negative ion FAB-MS/MS is a simple and useful technique for trace mixture analysis and structure elucidation of isoprenoid diphosphates.     

A new, model-free calculation method to determine the coordination modes and distribution of copper(II) among the metal binding sites of multihistidine peptides using circular dichroism spectroscopy

A new calculation method to determine microscopic protonation processes from CD spectra measured at different pH and Cu(II):ligand ratios was developed and used to give the relative binding strengths for the three histidines of hsPrP(84-114), a 31-mer polypeptide modeling the N-terminal copper(II) binding region of human (homo sapiens) prion protein. Mutants of hsPrP(84-114) with two or one histidyl residues have also been synthesized and their copper(II) complexes studied by CD spectroscopy. The 1-His models were analyzed first, and the molar CD spectra for the different coordination modes on the different histidines were calculated using the general computational program PSEQUAD. These spectra were deconvoluted into the sum of Gaussian curves and used as a first parameter set to calculate the molar spectra for the different coordination modes (3N and 4N coordination) and coordination positions (His85, His96 and His111) of the 2-His peptides. The calculation method therefore does not require the direct use of CD spectra measured in the smaller peptide models. This is a significant improvement over earlier calculation methods. In the same runs, the stepwise deprotonation pK(mic) values were refined and the pH-dependent distribution of copper(II) between the two histidines was determined. The results revealed the high, but different copper(II) binding affinities of the three separate histidines in the following order: His85 < His96His111. The calculation also showed that molar CD spectra which belong to the same coordination mode and coordination position in different ligands have very similar transition energies but different intensities. For this reason, direct transfer of molar CD spectra between different ligands may be a source of error, but the pK(mic) values and the copper(II) binding preferences are transferable from the 2-His peptides to the 3-His hsPrP(84-114).     

Oligopurine.oligopyrimidine tracts do not have the same conformation as analogous polypurine.polypyrimidines

The ability of tracts of synthetic oligopurine.oligopyrimidines containing both adenosine and guanosine residues to approach the conformation of analogous polypurine.polypyrimidines has been examined as a function of tract length by CD spectroscopy. Tracts of up to 19 contiguous, alternating dA and dG residues yield CD spectra that are distinctly different from that of the analogous alternating polymer. Thus the structural changes reflected in the unusual CD spectrum of poly[d(AG)].poly[d(CT)] must require even longer tract lengths. Tracts of contiguous adenosines flanked by guanosine residues were seen to approach the CD spectrum of poly[dA].poly[dT] quite slowly as a function of tract length, requiring more than 24 contiguous adenosines to give CD spectra similar to the homopolymer. These results lead us to the conclusion that oligopurine tracts in vivo are not well modeled by synthetic polypurine.polypyrimidines with one or two base pair repeating units.     

Circular dichroism calculations for double-stranded polynucleotides of repeating sequence

Optical property calculations are presented for poly(A·U), poly[(A-U)·(A-U)], poly(G·C), and poly[(G-C)·(G-C)] in RNA, B-DNA, and C-DNA conformations. An all-order classical coupled oscillator polarizability theory was used, and an effective dielectric constant of 2 was assumed. The calculated CD spectra were found to be sensitive to both geometry and sequence. Agreement with the measured CD spectra of poly(A·U), poly(G·C), and poly(dG·dC) is very good. Calculations for other sequences and geometries are less satisfactory and are particularly poor for poly[(G-C)·(G-C)] in RNA geometry and poly(A·T) in B-DNA geometry. Attempts to improve agreement with measured spectra by varying monomer properties have been only partially successful for these calculations, but they illustrate the types of changes that may prove to be necessary. Calculations using other published X-ray coordinates for certain deoxypolynucleotides of simple sequence, some of which are quite different from B-DNA coordinates, did not result in better agreement with measured spectra. Finally, the dependence of the calculated CD on chain length is examined. Results show that non-nearest neighbor interactions can be important when runs of 3 or more identical base pairs appear in a given sequence.     

Conformational diversity of acid-denatured cytochrome c studied by a matrix analysis of far-UV CD spectra.

The singular value decomposition (SVD) analysis was applied to a large set of far-ultraviolet circular dichroism (far-UV CD) spectra (100-400 spectra) of horse heart cytochrome c (cyt c). The spectra were collected at pH 1.7-5.0 in (NH4)2SO4, sorbitol and 2,2,2-trifluoroethanol (TFE) solutions. The present purpose is to develop a rigorous matrix method applied to far-UV CD spectra to resolve in details conformational properties of proteins in the non-native (or denatured) regions. The analysis established that three basis spectral components are contained in a data set of difference spectra (referred to the spectrum of the native state) used here. By a further matrix transformation, any observed spectrum could be decomposed into fractions of the native (N), the molten-globule (MG), the highly denatured (D), and the alcohol-induced helical (H) spectral forms. This method could determine fractional transition curves of each conformer as a function of solution conditions, which gave the results consistent with denaturation curves of cyt c monitored by other spectroscopic methods. The results in sorbitol solutions, for example, suggested that the preferential hydration effect of the co-solvent stabilizes the MG conformer of cyt c. This report has found that the systematic SVD analysis of the far-UV CD spectra is a powerful tool for the conformational analysis of the non-native species of a protein when it is suitably supplemented with other experimental methods.     

Circular dichroic spectroscopy of membrane haemoproteins. The molecular determinants of the dichroic properties of the b cytochromes in various ubiquinol:cytochrome c reductases

The circular dichroism (CD) of dihaem cytochrome b from mitochondrial and bacterial ubiquinol:cytochrome-c reductase (bc1 complex) has been characterized. The dichroic properties of the yeast purified cyt b are very similar to those of the native cyt b within the mitochondrial bc1 complex. The CD spectra in the Soret region of the native cytochrome b present in all species studied show an intense bisignate Cotton effect having a zero-crossing wavelength close to the absorbance maximum. In preparations partially or completely depleted of the low-potential b haem (b1) the CD spectra exhibit a single positive Cotton effect resembling the corresponding absorption spectrum. This is particularly evident in the purified cytochrome b-562 from Rhodobacter sphaeroides R26, which contains only the high-potential b haem (bh). These spectral features together with the reconstitution of the cytochrome b1 haem have been used to resolve the CD contribution of each haem to the CD spectra of cytochrome b. The mechanisms which might be responsible for the optical activity have been examined. It appears that the CD spectra of cytochrome b derive from both the mutual interaction of its two haems (giving rise to exciton coupling) and to the interaction of each haem with nearby aromatic residues, other than the pairs of histidines which coordinate the iron. The dipole coupling between haem and aromatic residues appears to be more important than exciton coupling in the CD spectra of oxidized b cytochromes and correlations have been made between the CD features and the proposed structure of cytochrome b.     

Analysis of the circular dichroism spectrum of proteins using the convex constraint algorithm: a practical guide.

Due to the time scale of circular dichroism (CD) measurements, it is theoretically possible to deconvolute such a spectrum if the pure CD spectra differ significantly from one another. In the last decade several methods have been published aiming at obtaining the conformational weights, or percentages (which are the coefficients for a linear combination) of the so-called typical secondary structural elements making up the three-dimensional structure of proteins. Two methods that can be used to determine the secondary structures of proteins are described here. The first method, called LINCOMB, is a simple algorithm based on a least-squares fit with a set of reference spectra representing the known secondary structures and yielding an estimation of weights attributed to alpha-helix, beta-pleated sheet (mainly antiparallel), beta-turns, unordered form, and aromatic/disulfide (or nonpeptide) contributions of the protein being analyzed. This method requires a "template" or reference curve set, which was obtained from the second method. The second method, "convex constraint analysis," is a general deconvolution method for a CD spectra set of any variety of conformational type. The algorithm, based on a set of three constraints, is able to deconvolute a set of CD curves to its common "pure"-component curves and conformational weights. To analyze a single CD spectrum with this method, the spectrum is appended to the data set used as a reference data set. As a way to determine the reliability of the algorithm and provide a guideline to its usage, some applications are presented.     

On-line monitoring of nine different batch cultures of E. coli by mid-infrared spectroscopy, using a single spectra library for calibration

A single spectra library was used to monitor on-line, by mid-infrared spectroscopy, nine different batch cultures of Escherichia coli performed with various medium compositions, including chemically complex formulations. Whereas the classic chemometrics approach would have required the preparation and measurement of hundreds of standards, only six spectra were included in the library. These included the molar absorbance of the four main metabolites (i.e. glucose, glycerol, ammonium and acetate), and the remaining two were drift spectra found by factor analysis. The accuracy of the prediction was not altered by a change of the carbon source, the ammonium concentration or even the addition of chemically undefined compounds, such as yeast extract and peptone. The standard errors of prediction averaged over the nine experiments were 8.0, 12.3, 5.9 and 5.6 mM for glucose, glycerol, ammonium and acetate, respectively. Inclusion of two drift spectra in the library provided an estimation of how noisy an experiment was. This also allowed detection of batch cultures that require further investigation, namely runs which were subject to large signal drift or during which an unexpected compound was produced, without having to carry out time-consuming off-line analyses.     

Novel circular dichroism spectroscopic approach for detection of ligand binding of proteins: Avidin as example

Various globular proteins having low or no α-helical content exhibit atypical far-ultraviolet (UV) circular dichroism (CD) spectra due to aromatic-aromatic residue and aromatic residue-peptide bond exciton coupling interactions. As a representative example of such proteins, far-UV CD spectra of chicken avidin were recorded before and after the addition of different small molecules. Intensity increase-decrease and/or wavelength shift of the positive CD peak of avidin at 228 nm were observed in the presence of various drugs, dyes, and natural compounds. The results were interpreted by exciton interactions between the aromatic residues of the biotin binding site and the substances bound to it. This novel, fast, microgram (μg) scale approach can be applied for detection of ligand binding of additional proteins displaying avidin-like far-UV CD spectra.     

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5′-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5′-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the K_i value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11302-021-09802-w.     

Estimating analytical variability in two-dimensional data

Throughout the course of drug development there are many instances in which a variability assessment within a set of analytical data is required, which may be challenging for techniques that produce two-dimensional data. This note describes an interval-based approach to variability assessment and demonstrates its applicability for analysis of near-UV circular dichroism (CD) spectra. The approach is generalizable and could be applied to two-dimensional data from other analytical techniques as well.