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.     

Differentiation between transmembrane helices and peripheral helices by the deconvolution of circular dichroism spectra of membrane proteins.

The interpretation of the circular dichroism (CD) spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as X-ray or NMR data. Therefore, these methods are inappropriate for a CD database whose secondary structures are unknown, as in the case of the membrane proteins. The convex constraint analysis algorithm (Perczel, A., Hollósi, M., Tusnády, G., & Fasman, G. D., 1991, Protein Eng. 4, 669-679), on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived "pure" CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of alpha helices (the alpha helix in the soluble domain and the alpha T helix, for the transmembrane alpha helix), a beta-pleated sheet, a class C-like spectrum related to beta turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the alpha T helix was characterized by a positive red-shifted band in the range 195-200 nm (+95,000 deg cm2 dmol-1), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222-nm band (-50,000 and -60,000 deg cm2 dmol-1, respectively) in comparison with the regular alpha helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of +70,000, -30,000, and -30,000 deg cm2 dmol-1, respectively.     

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.     

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.     

Frequency analysis of infrared absorption and vibrational circular dichroism of proteins in D2O solution.

The IR absorption frequencies as derived from second derivatives of the Fourier transform IR spectra of the amide I' bands of globular proteins in D2O are compared to those obtained from band fitting of the vibrational circular dichroism (VCD) spectra. The two sets of frequencies are in very good agreement, yielding consistent ranges where amide I' VCD and IR features occur. Use of VCD to complement the IR allows one to add sign information to the frequency information so that features occurring in the overlapping frequency ranges that might arise from different secondary structures can be better discriminated. From this comparison, it is clear that correlation just of the frequency of a given IR transition to secondary structure can lead to a nonunique solution. Different sign patterns were identified for correlated groups of globular proteins in restricted frequency ranges that have been previously assigned to defined secondary structural elements. Hence, different secondary structural elements must contribute band components to a given frequency range.     

Circular dichroism analyses of membrane proteins: an examination of differential light scattering and absorption flattening effects in large membrane vesicles and membrane sheets

The circular dichroism spectra of membrane suspensions are distorted by differential light scattering and absorption flattening effects, which arise as a consequence of the large size of the membrane particles relative to the wavelength of light and the high concentration of proteins in the membranes. In this paper, the consequences of these phenomena on the protein spectra of large membrane particles are discussed, and methods for eliminating them are examined. The distortions due to differential light scattering are relatively small in membrane systems, and can be compensated for by use of a large detector acceptance angle geometry. Several methods for correcting for differential flattening, which introduces a substantial distortion, have been evaluated, and a new method, the flattening quotient approach, which produces by far the best results, is described. Since the secondary structures calculated from circular dichroism spectra are highly dependent on accurate spectral shape and magnitude, this method for correcting the spectra may find general application in circular dichroism studies of membrane proteins.     

Vacuum-ultraviolet circular dichroism analysis of biomolecules

The vacuum-ultraviolet circular dichroism (VUVCD) spectra of various amino acids, saccharides, and proteins were measured using a synchrotron-radiation CD spectrophotometer at HiSOR/HSRC that is capable of measuring the CD spectra down to 140 nm in aqueous solution. L-Isomers of amino acids show two successive positive peaks at around 200 and 180 nm depending on the side chain. The ab initio assignment by time-dependent density functional theory predicts that these peaks are attributed to n-pi* and pi-pi* transitions of the carboxyl group, respectively. Most mono- and disaccharides exhibit characteristic peaks at around 170 nm, sensitively depending on the anomeric and axial/equatorial configurations of hydroxyl groups, trans-gauche conformations of the hydroxymethyl group, and the type of glycosidic linkage. The VUVCD spectra of 31 globular proteins allow us to estimate more accurately the content and number of alpha-helix and beta-strand segments by extending the short-wavelength limit of the analytical program SELCON3 down to 160 nm. These results demonstrate that synchrotron-radiation VUVCD spectroscopy is a useful tool for structure analyses of biomolecules in solution based on the higher energy transitions of chromophores.     

Conformational flexibility of a scorpion toxin active on mammals and insects: a circular dichroism study

Three scorpion toxins have been analyzed by circular dichroism in water and in 2,2,2-trifluoroethanol (TFE) solutions. These toxins were chosen because they are representative of three kinds of pharmacological activities: (1) toxin AaH IT2, an antiinsect toxin purified from the venom of Androctonus australis Hector, which is able to bind to insect nervous system preparation, (2) toxin Css II, from the venom of Centruroides suffusus suffusus, which is a beta-type antimammal toxin capable of binding to mammal nervous system preparation, and (3) the toxin Ts VII from the venom of Tityus serrulatus, which is able to bind to both types of nervous systems. In order to minimize bias, CD data were analyzed by a predictive algorithm to assess secondary structure content. Among the three molecules, Ts VII presented the most unordered secondary structure in water, but it gained in ordered forms when solubilized in TFE. These results indicated that the Ts VII backbone is the most flexible, which might result in a more pronounced tendency for this toxin molecule to undergo conformational changes. This is consistent with the fact that it competes with both antiinsect and beta-type antimammal toxins for the binding to the sodium channel.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Rationalizing alpha-helical membrane protein crystallization

X-ray crystallography is currently the most successful method for determining the three-dimensional structure of membrane proteins. Nevertheless, growing the crystals required for this technique presents one of the major bottlenecks in this area of structural biology. This is especially true for the alpha-helical type membrane proteins that are of particular interest due to their medical relevance. To address this problem we have undertaken a detailed analysis of the crystallization conditions from 121 alpha-helical membrane protein structures deposited in the Protein Data Bank. This information has been analyzed so that the success of different parameters can be easily compared for different membrane protein families. Concurrent with this analysis, we also present the new sparse matrix crystallization screen MemGold.     

Deciphering membrane protein structures from protein sequences

ABSTRACT: Co-evolving positions within protein sequences have been used as spatial constraints to develop a computational approach for modeling membrane protein structures.     

DichroWeb,a website for calculating protein secondary structure from circular dichroism spectroscopic data

Circular dichroism (CD) spectroscopy is a widely‐used method for characterizing the secondary structures of proteins. The well‐established and highly used analysis website, DichroWeb (located at: http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) enables the facile quantitative determination of helix, sheet, and other secondary structure contents of proteins based on their CD spectra. DichroWeb includes a range of reference datasets and algorithms, plus graphical and quantitative methods for determining the quality of the analyses produced. This article describes the current website content, usage and accessibility, as well as the many upgraded features now present in this highly popular tool that was originally created nearly two decades ago.     

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.     

Conformational analysis of hemopexin by fourier-transform infrared and circular dichroism spectroscopy

Hemopexin is a serum glyco-protein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptormediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% β-structure, 15% α-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme–hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the hemeprotein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis. © 1994 Wiley-Liss, Inc.     

Application of SVM to predict membrane protein types

As a continuous effort to develop automated methods for predicting membrane protein types that was initiated by Chou and Elrod (PROTEINS: Structure, Function, and Genetics, 1999, 34, 137-153), the support vector machine (SVM) is introduced. Results obtained through re-substitution, jackknife, and independent data set tests, respectively, have indicated that the SVM approach is quite a promising one, suggesting that the covariant discriminant algorithm (Chou and Elrod, Protein Eng. 12 (1999) 107) and SVM, if effectively complemented with each other, will become a powerful tool for predicting membrane protein types and the other protein attributes as well.     

Improved sequence-based prediction of protein secondary structures by combining vacuum-ultraviolet circular dichroism spectroscopy with neural network

Synchrotron-radiation vacuum-ultraviolet circular dichroism (VUVCD) spectroscopy can significantly improve the predictive accuracy of the contents and segment numbers of protein secondary structures by extending the short-wavelength limit of the spectra. In the present study, we combined VUVCD spectra down to 160 nm with neural-network (NN) method to improve the sequence-based prediction of protein secondary structures. The secondary structures of 30 target proteins (test set) were assigned into alpha-helices, beta-strands, and others by the DSSP program based on their X-ray crystal structures. Combining the alpha-helix and beta-strand contents estimated from the VUVCD spectra of the target proteins improved the overall sequence-based predictive accuracy Q(3) for three secondary-structure components from 59.5 to 60.7%. Incorporating the position-specific scoring matrix in the NN method improved the predictive accuracy from 70.9 to 72.1% when combining the secondary-structure contents, to 72.5% when combining the numbers of segments, and finally to 74.9% when filtering the VUVCD data. Improvement in the sequence-based prediction of secondary structures was also apparent in two other indices of the overall performance: the correlation coefficient (C) and the segment overlap value (SOV). These results suggest that VUVCD data could enhance the predictive accuracy to over 80% when combined with the currently best sequence-prediction algorithms, greatly expanding the applicability of VUVCD spectroscopy to protein structural biology.     

Estimation of protein secondary structure and error analysis from circular dichroism spectra

The estimation of protein secondary structure from circular dichroism spectra is described by a multivariate linear model with noise (Gauss-Markoff model). With this formalism the adequacy of the linear model is investigated, paying special attention to the estimation of the error in the secondary structure estimates. It is shown that the linear model is only adequate for the alpha-helix class. Since the failure of the linear model is most likely due to nonlinear effects, a locally linearized model is introduced. This model is combined with the selection of the estimate whose fractions of secondary structure summate to approximately one. Comparing the estimation from the CD spectra with the X-ray data (by using the data set of W.C. Johnson Jr., 1988, Annu. Rev. Biophys. Chem. 17, 145-166) the root mean square residuals are 0.09 (alpha-helix), 0.12 (anti-parallel beta-sheet), 0.08 (parallel beta-sheet), 0.07 (beta-turn), and 0.09 (other). These residuals are somewhat larger than the errors estimated from the locally linearized model. In addition to alpha-helix, in this model the beta-turn and "other" class are estimated adequately. But the estimation of the antiparallel and parallel beta-sheet class remains unsatisfactory. We compared the linear model and the locally linearized model with two other methods (S. W. Provencher and J. Gl?ckner, 1981, Biochemistry 20, 1085-1094; P. Manavalan and W. C. Johnson Jr., 1988, Anal. Biochem. 167, 76-85). The locally linearized model and the Provencher and Gl?ckner method provided the smallest residuals. However, an advantage of the locally linearized model is the estimation of the error in the secondary structure estimates.     

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.     

Secondary structural analysis of retrovirus integrase: characterization by circular dichroism and empirical prediction methods

The retrovirus integrase (IN) protein is essential for integration of viral DNA into host DNA. The secondary structure of the purified IN protein from avian myeloblastosis virus was investigated by both circular dichroism (CD) spectroscopy and five empirical prediction methods. The secondary structures determined from the resolving of CD spectra through a least-squares curve fitting procedure were compared with those predicted from four statistical methods, e.g., the Chou-Fasman, Garnier-Osguthorpe-Robson, Nishikawa-Ooi, and a JOINT scheme which combined all three of these methods, plus a pure a priori one, the Ptitsyn-Finkelstein method. Among all of the methods used, the Nishikawa-Ooi prediction gave the closest match in the composition of secondary structure to the CD result, although the other methods each correctly predicted one or more secondary structural group. Most of the alpha-helix and beta-sheet states predicted by the Ptitsyn-Finkelstein method were in accord with the Nishikawa-Ooi method. Secondary structural predictions by the Nishikawa-Ooi method were extended further to include IN proteins from four phylogenetic distinct retroviruses. The structural relationships between the four most conserved amino acid blocks of these IN proteins were compared using sequence homology and secondary structure predictions.