CD studies on films of amyloid proteins and polypeptides: quantitative g-factor analysis indicates a common folding motif

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a "cross-beta" structure, with extended beta-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P. McPhie, Analytical Biochemistry, 2001, Vol. 293, pp. 109-119). Amyloid films of Abeta-(1-40) peptide, beta-2-microglobulin, insulin, and three homopolypeptides show very intense CD spectra, compatible with the presence of a beta-helix-like structure, arranged in a common framework in the fibrils. The extent of this structure was estimated as 45-80% in the protein fibrils and 30-80% in the polypeptide fibrils.     

Secondary structure analyses of protein films on gold surfaces by circular dichroism

In order to analyze the secondary structures of protein molecules adsorbed on gold surfaces, circular dichroism (CD) spectra were measured and the secondary structure contents of protein ultra-thin films were estimated quantitatively. A disulfide group was introduced to cytochrome b(562) (cyt.b562), which is a water-soluble b-type heme protein. The cyt.b562 molecules self-assembled to form an ultra-thin protein film both on a gold substrate modified with 2,2(')-dithiodiacetic acid and on a bare gold surface. CD measurements were carried out both in solution and in air, and these results were compared. The protein denaturation was partially prevented, not only in solution but also in air, by both the modification of the substrate and the introduction of the anchor group to the protein molecule. The secondary structure contents of ultra-thin protein films on flat gold surfaces were observed for the first time both in solution and in air by CD spectra.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Overestimated accuracy of circular dichroism in determining protein secondary structure

Circular dichroism (CD) is a spectroscopic technique widely used for estimating protein secondary structures in aqueous solution, but its accuracy has been doubted in recent work. In the present paper, the contents of nine globular proteins with known secondary structures were determined by CD spectroscopy and Fourier transform infrared spectroscopy (FTIR) in aqueous solution. A large deviation was found between the CD spectra and X-ray data, even when the experimental conditions were optimized. The content determined by FTIR was in good agreement with the X-ray crystallography data. Therefore, CD spectra are not recommended for directly calculating the content of a protein’s secondary structure.     

Circular dichroism of films of polynucleotides

The CD spectra of films of the lithium salt of E. coli and calf thymus DNA, and alternating d-AT : AT were measured as a function of relative humidity. Films of the ammonium acetate salt of DNA were also measured. The ammonium films yield the previously reported A-form CD spectra. A possible explanation for the small magnitude of the 260-nm band of the A-form film spectra compared to double-stranded RNA spectra is that the film DNA is in a different conformation than RNA within the A family of conformations. At relative humidities of 92% or lower, a negative nonconservative CD spectrum with negative minima near 270 and 210 nm is observed with the lithium films. The magnitude of the minima varies from film to film. In films of DNA the magnitude ranges from a delta epsilon of ?5 to ?35; d-AT : AT films show magnitudes to ?300. CD spectra of this type are designated Ψ spectra. Similar spectra have been reported from reconstituted complexes of DNA and polylysine or f-1 histone. If the origins of the film and protein–DNA complex spectra are similar, the complex spectra are not the result of specific secondary structural changes induced in the DNA by the protein fraction. Theoretical analysis suggests that Ψ spectra are not the result of changes in the secondary or tertiary structure of DNA. Instead, the previously proposed explanation based on liquid crystals is favored. The DNA could form asymmetric structures with long-range periodicity. It is likely that the observed CD spectra of f-1 complexes are artifacts of DNA aggregation. The possibility that some other previously published spectra of protein–DNA complexes also reflect artifacts is suggested.     

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.     

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.     

A reference database for circular dichroism spectroscopy covering fold and secondary structure space

MOTIVATION: Circular Dichroism (CD) spectroscopy is a long-established technique for studying protein secondary structures in solution. Empirical analyses of CD data rely on the availability of reference datasets comprised of far-UV CD spectra of proteins whose crystal structures have been determined. This article reports on the creation of a new reference dataset which effectively covers both secondary structure and fold space, and uses the higher information content available in synchrotron radiation circular dichroism (SRCD) spectra to more accurately predict secondary structure than has been possible with existing reference datasets. It also examines the effects of wavelength range, structural redundancy and different means of categorizing secondary structures on the accuracy of the analyses. In addition, it describes a novel use of hierarchical cluster analyses to identify protein relatedness based on spectral properties alone. The databases are shown to be applicable in both conventional CD and SRCD spectroscopic analyses of proteins. Hence, by combining new bioinformatics and biophysical methods, a database has been produced that should have wide applicability as a tool for structural molecular biology.     

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.     

Secondary structures of proteins adsorbed onto aluminum hydroxide: infrared spectroscopic analysis of proteins from low solution concentrations

Comparative studies of the secondary structures of six model proteins, adsorbed onto aluminum hydroxide gel (Alhydrogel) or in aqueous solution, were carried out by Fourier transform infrared (FTIR) spectroscopy. The analysis of high-quality spectra of all six model proteins, with a broad range of secondary structure compositions, obtained at 15 mg/ml by the conventional method and at 0.5 and 1.0 mg/ml adsorbed to Alhydrogel revealed that adsorption onto hydrophilic surfaces of aluminum hydroxide particles did not alter the secondary structures of the proteins. The results of this study suggest that adsorbing proteins to Alhydrogel provides a means of obtaining FTIR spectra to study secondary structure and conformational changes of proteins in aqueous solution at very low concentrations. The new procedure effectively lowers the concentration requirement for FTIR studies of proteins in aqueous solutions by at least 40-fold, as compared with the conventional FTIR method. It permits FTIR study of proteins to be carried out in the same concentration range as is used for circular dichroism and fluorescence, thereby making it possible to compare structural information obtained by three commonly used techniques in protein biophysical characterization.     

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.     

Protein secondary structure from Fourier transform infrared spectroscopy: a data base analysis

An infrared (ir) method to determine the secondary structure of proteins in solution using the amide I region of the spectrum has been devised. The method is based on the circular dichroism (CD) matrix method for secondary structure analysis given by Compton and Johnson (L. A. Compton and W. C. Johnson, 1986, Anal. Biochem. 155, 155-167). The infrared data matrix was constructed from the normalized Fourier transform infrared spectra from 1700 to 1600 cm-1 of 17 commercially available proteins. The secondary structure matrix was constructed from the X-ray data of the seventeen proteins with secondary structure elements of helix, beta-sheet, beta-turn, and other (random). The CD and ir methods were compared by analyzing the proteins of the CD and ir databases as unknowns. Both methods produce similar results compared to structures obtained by X-ray crystallographic means with the CD slightly better for helix conformation, and the ir slightly better for beta-sheet. The relatively good ir analysis for concanavalin A and alpha-chymotrypsin indicate that the ir method is less affected by the presence of aromatic groups. The concentration of the protein and the cell path length need not be known for the ir analysis since the spectra can be normalized to the total ir intensity in the amide I region. The ir spectra for helix, beta-sheet, beta-turn, and other, as extracted from the data-base, agree with the literature band assignments. The ir data matrix and the inverse matrix necessary to analyze unknown proteins are presented.     

Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid beta(Abeta) and alpha-synuclein fragment peptide (NAC).

The effects of oligopeptides on the secondary structures of Abeta and NAC, a fragment of alpha-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of Abeta or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at Abeta(17-21), had no effect on the secondary structures of Abeta(1-28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of Abeta(1-28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the alpha-helical content, while it increased the beta-sheet content of Abeta(1-28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of Abeta(1-28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a beta-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21-22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the beta-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution. Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time.     

Vibrational circular dichroism of polypeptides. III. Film studies of several alpha-helical and beta-sheet polypeptides

Vibrational CD (VCD) of amides A, I, and II vibrations of a variety of polypeptide films have been measured. VCD of films of α-helical and β-sheet structures are compared in the three regions. Reproducible spectra could only be obtained for thin films free of orientation dependence. The sign and band shape of the VCD of films are not always the same as that in solution. However, the magnitude of the observed VCD seems to correlate with the secondary structure such that α-helical molecules typically have much larger Δε/ε values than do β-sheet molecules. The possibility of interference by artifacts owing to light-scattering effects is discussed.     

Estimation of protein secondary structure from circular dichroism spectra: inclusion of denatured proteins with native proteins in the analysis

We have expanded our reference set of proteins used in the estimation of protein secondary structure by CD spectroscopy from 29 to 37 proteins by including 3 additional globular proteins with known X-ray structure and 5 denatured proteins. We have also modified the self-consistent method for analyzing protein CD spectra, SELCON3, by including a new selection criterion developed by W. C. Johnson, Jr. (Proteins Struct. Funct. Genet. 35, 307-312, 1999). The secondary structure corresponding to the denatured proteins was approximated to be 90% unordered, owing to the spectral similarity of the denatured proteins and unordered structures. We examined the thermal denaturation of ribonuclease T1 by CD using both the original and expanded sets of reference proteins and obtained more consistent results with the expanded set. The expanded set of reference proteins will be helpful for the determination of protein secondary structure from protein CD spectra with higher reliability, especially of proteins with significant unordered structure content and/or in the course of denaturation.     

Local structures in unfolded lysozyme and correlation with secondary structures in the native conformation: helix-forming or -breaking propensity of peptide segments

CD spectra of reduced and S-3-(trimethylated amino) propylated lysozyme (TMAP lysozyme) have been measured in various solutions containing guanidine hydrochloride or trifluoroethanol (TFE). The CD spectra indicate that there remain residual secondary structures in protein in aqueous solution. The addition of TFE further promotes the formation of secondary structures. In order to examine whether secondary structures are evenly induced over all the polypeptide chain, or locally at particular segments, the limited proteolysis of TMAP lysozyme by trypsin has been performed, and the CD spectra of all the final and intermediate products have been observed in solutions containing TFE. As a result, the fragments vary in a helix-forming propensity. The CD spectra of peptide fragments T5, T7, T9T10, T12T13, T14T15T16, and T17T18 are not significantly affected by the addition of TFE, where T refers to the nomenclature of R.E. Canfield [(1963), Journal of Biological Chemistry, Vol. 238, pp. 2691-2697]. They are fragments of a helix-breaking propensity. On the other hand, fragment I2 composed of T1-T4, and fragments T6T7, T8, and T11, attain secondary structures with the addition of TFE. They are fragments of a helix-forming propensity. Further, it is found that the fragments of a helix-forming propensity just correspond to the helical segments in native lysozyme. We examine the interactions between neighboring fragments, which contribute to the stabilization of local structures along the polypeptide chain.     

Chemometric tools for classification and elucidation of protein secondary structure from infrared and circular dichroism spectroscopic measurements

Protein classification and characterization often rely on the information contained in the protein secondary structure. Protein class assignment is usually based on X-ray diffraction measurements, which need the protein in a crystallized form, or on NMR spectra, to obtain the structure of a protein in solution. Simple spectroscopic techniques, such as circular dichroism (CD) and infrared (IR) spectroscopies, are also known to be related to protein secondary structure, but they have seldom been used for protein classification. To see the potential of CD, IR, and combined CD/IR measurements for protein classification, unsupervised pattern recognition methods, Principal Component Analysis (PCA) and cluster analysis, are proposed first to check for natural grouping tendencies of proteins according to their measured spectra. Partial Least Squares Discriminant Analysis (PLS-DA), a supervised pattern recognition method, is used afterwards to test the possibility to model explicitly each protein class and to test these models in class assignment of unknown proteins. Determination of the protein secondary structure, understood as the prediction of the abundance of the different secondary structure motifs in the biomolecule, was carried out with the local regression method interval Partial Least Squares (iPLS). CD, IR, and CD/IR measurements were correlated to the fraction of the motif to be predicted, determined from X-ray measurements. iPLS builds models extracting the spectral information most correlated to a specific secondary motif and avoids the use of irrelevant spectral regions. Spectral intervals chosen by iPLS models provide structural information which can be used to confirm previous biochemical assignments or identify new motif-related spectral features. The predictive ability of the models built with the selected spectral regions has a quality similar to previous classical approaches.     

Spectral magnitude effects on the analyses of secondary structure from circular dichroism spectroscopic data

The effects of spectral magnitude on the calculated secondary structures derived from circular dichroism (CD) spectra were examined for a number of the most commonly used algorithms and reference databases. Proteins with different secondary structures, ranging from mostly helical to mostly beta-sheet, but which were not components of existing reference databases, were used as test systems. These proteins had known crystal structures, so it was possible to ascertain the effects of magnitude on both the accuracy of determining the secondary structure and the goodness-of-fit of the calculated structures to the experimental data. It was found that most algorithms are highly sensitive to spectral magnitude, and that the goodness-of-fit parameter may be a useful tool in assessing the correct scaling of the data. This means that parameters that affect magnitude, including calibration of the instrument, the spectral cell pathlength, and the protein concentration, must be accurately determined to obtain correct secondary structural analyses of proteins from CD data using empirical methods.     

Prediction of protein secondary structure from circular dichroism using theoretically derived spectra

Circular dichroism (CD) is a spectroscopic technique commonly used to investigate the structure of proteins. Major secondary structure types, alpha‐helices and beta‐strands, produce distinctive CD spectra. Thus, by comparing the CD spectrum of a protein of interest to a reference set consisting of CD spectra of proteins of known structure, predictive methods can estimate the secondary structure of the protein. Currently available methods, including K2D2, use such experimental CD reference sets, which are very small in size when compared to the number of tertiary structures available in the Protein Data Bank (PDB). Conversely, given a PDB structure, it is possible to predict a theoretical CD spectrum from it. The methodological framework for this calculation was established long ago but only recently a convenient implementation called DichroCalc has been developed. In this study, we set to determine whether theoretically derived spectra could be used as reference set for accurate CD based predictions of secondary structure. We used DichroCalc to calculate the theoretical CD spectra of a nonredundant set of structures representing most proteins in the PDB, and applied a straightforward approach for predicting protein secondary structure content using these theoretical CD spectra as reference set. We show that this method improves the predictions, particularly for the wavelength interval between 200 and 240 nm and for beta‐strand content. We have implemented this method, called K2D3, in a publicly accessible web server at http://www. ogic.ca/projects/k2d3 . Proteins 2012. © 2011 Wiley Periodicals, Inc.     

Molecular crowding inhibits intramolecular breathing motions in proteins

In aqueous solution some proteins undergo large-scale movements of secondary structures, subunits or domains, referred to as protein “breathing”, that define a native-state ensemble of structures. These fluctuations are sensitive to the nature and concentration of solutes and other proteins and are thereby expected to be different in the crowded interior of a cell than in dilute solution. Here we use a combination of wide angle X-ray scattering (WAXS) and computational modeling to derive a quantitative measure of the spatial scale of conformational fluctuations in a protein solution. Concentration-dependent changes in the observed scattering intensities are consistent with a model of structural fluctuations in which secondary structures undergo rigid-body motions relative to one another. This motion increases with decreasing protein concentration or increasing temperature. Analysis of a set of five structurally and functionally diverse proteins reveals a diversity of kinetic behaviors. Proteins with multiple disulfide bonds exhibit little or no increase in breathing in dilute solutions. The spatial extent of structural fluctuations appears highly dependent on both protein structure and concentration and is universally suppressed at very high protein concentrations.