The secondary structure of bacteriorhodopsin determined by Raman and circular dichroism spectroscopy

The secondary structure of bacterio-opsin (BO), the retinal free protein-component of bacteriorhodopsin (BR), has been determined by Raman spectroscopy. Additional circular dichroism (CD) measurements have revealed only negligible conformational differences between BO in apomembranes and BR in purple membranes. Therefore, the secondary structure of BR was derived from the Raman data of BO. The protein conformation was determined to consist of 72-82% helices, 2-11% beta-strands, and 11-17% beta-turns. Only about half of the helical structures correspond to alpha 1-helices, the other half possess non-alpha 1-helical structures. According to the analysis of the Raman data, the derived secondary structure of BR was obtained with high reliability for all structure classes which can be distinguished by this method within the given uncertainty range. This is a remarkable difference from recently published secondary structural data derived from CD measurements where the helix content was reported to be between 50 and 80%. The inherent experimental and methodological uncertainties of the CD-technique leading to such a range of variation are critically discussed in comparison to the method of Raman spectroscopy. The combined application of Raman and CD spectroscopy, as performed here, is demonstrated to be a substantial improvement in the secondary structure determination of retinal-containing membrane proteins. On the basis of our results, some of the recently proposed structural models of BR with a beta-strand content of more than 11% can be ruled out.     

Structural features of human leukocyte interferon A as determined by circular dichroism spectroscopy

The solution structure of human leukocyte (alpha) interferon-A (LeIF-A) purified from E. coli extracts was investigated by circular dichroism spectroscopy. At pH 8.2, the native molecule exhibits 40-70% alpha-helix and no clearly detectable beta-structure. The tertiary structure includes a closely-packed interior containing at least one tryptophan side-chain. Titration to pH 1.5 produces a partial loss of structural features which are rapidly regained on return to neutral pH.     

The secondary structure of ovomucoid and its domains as studied by circular dichroism

The secondary structure of chicken egg white ovomucoid and its domains was studied by means of circular dichroism (CD). The various domains (domain I, domains II · III, domains I · II, and domain III with and without carbohydrate) were prepared from the ovomucoid by cyanogen bromide cleavage and Staphylococcus aureus protease V-8 digestions. The carbohydrate moiety in the ovomucoid was isolated after its extensive pronase and endo-β-N-acetylglucosaminidase H digestions. On the net CD spectra of polypeptide chain in the ovomucoid and domain preparations, which were obtained by subtracting the spectrum of the carbohydrate moiety from their spectra, the fractions of secondary structure were calculated by the method of Chang et al. (Chang, C.T., Wu, C.S.C. and Yang, J.T. (1978) Anal. Biochem. 91, 13–31). The estimated composition of the secondary structure in the ovomucoid was as follows: α-helix, 26%; β-structure, 46%; β-turn, 10%; and random coil, 18%. The secondary structure compositions estimated for the three domains suggested that domains I and II were homologous to one another but not to domain III in regard to the proportion of secondary structure content.     

gamma-Zein secondary structure in solution by circular dichroism

The proline-rich N-terminal domain of gamma-zein has been reported in relevant processes, which include its ability to cross the cell membranes. Evidences indicate that synthetic hexapeptide (PPPVHL), naturally found in N-terminal portion of gamma-zein, can adopt the polyproline II (PPII) conformation in aqueous solution. The secondary structure of gamma-zein in maize protein bodies had been analyzed by solid state Fourier transform infrared and nuclear magnetic resonance spectroscopies. However, it was not possible to measure PPII content in physiological environment since the beta-sheet and PPII signals overlap in both solid state techniques. Here, the secondary structure of gamma-zein has been analyzed by circular dichroism in SDS aqueous solution with and without ditiothreitol (DTT), and in 60% of 2-propanol and water with DTT. The results show that gamma-zein has high helical content in all solutions. The PPII conformation was present at about 7% only in water/DTT solution.     

Conformational structure of bombesin as studied by vibrational and circular dichroism spectroscopy

Raman and Fourier transform infrared (FTIR) spectroscopies and circular dichroism (CD) have been applied to investigate the secondary structure of bombesin in the solid state and in phosphate buffer solution (pH 3.8). At concentrations around 10⁻⁵ M, circular dichroism reveals that bombesin exists as an irregular or disordered conformation. However, the secondary structure of the peptide appears to be a mixture of disordered structure and intermolecular β-sheets in 0.01 M sodium phosphate buffer when the peptide concentrations are higher than around 6.5 mM. The tendency of bombesin to form aggregated β-sheet species seems to be originated mainly in the sequence of the residues 7–14, as supported by the Raman spectra and β-sheet propensities (P_β) of the amino-acid residues. It is the hydrophobic force of this amino-acid sequence, and not a salt bridge effect, that is the factor responsible for the formation of peptide aggregates.     

Going deep into protein secondary structure with synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy is a fast, powerful, well-established, and widely used analytical technique in the biophysical and structural biology community to study protein secondary structure and to track changes in protein conformation in different environments. The use of the intense light of a synchrotron beam as the light source for collecting CD measurements has emerged as an enhanced method, known as synchrotron radiation circular dichroism (SRCD) spectroscopy, that has several advantages over the conventional CD method, including a significant spectral range extension for data collection, deeper access to the lower limit (cut-off) of conventional CD spectroscopy, an improved signal-to-noise ratio to increase accuracy in the measurements, and the possibility to collect measurements in highly absorbing solutions. In this review, we discuss different applications of the SRCD technique by researchers from Latin America. In this context, we specifically look at the use of this method for examining the secondary structure and conformational behavior of proteins belonging to the four main classes of the hierarchical protein domain classification CATH (Class, Architecture, Topology, Homology) database, focusing on the advantages and improvements associated with SRCD spectroscopy in terms of characterizing proteins composed of different structural elements.     

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.     

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.     

Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases

Circular dichroism (CD) spectroscopy has been a valuable method for the analysis of protein secondary structures for many years. With the advent of synchrotron radiation circular dichroism (SRCD) and improvements in instrumentation for conventional CD, lower wavelength data are obtainable and the information content of the spectra increased. In addition, new computation and bioinformatics methods have been developed and new reference databases have been created, which greatly improve and facilitate the analyses of CD spectra. This article discusses recent developments in the analysis of protein secondary structures, including features of the DICHROWEB analysis webserver.     

beta-sheet structure formation of proteins in solid state as revealed by circular dichroism spectroscopy

Cross beta-sheet structure formation and abnormal aggregation of proteins are thought to be pathological characteristics of some neurodegenerative disorders. To investigate the novel structural transformation and aggregation, the solid-state secondary structures of some proteins and peptides associated in thin films were determined by circular dichroism spectroscopy. Insulin, lysozyme, DsbA protein, luciferase, and ovalbumin peptide fall into one group; they show no or slight structural rearrangement from solution to the solid state. Another group, including bovine serum albumin, ovalbumin, alpha-synuclein, and plasminogen activator inhibitor-1 (PAIRC) peptide, undergo structural transformation with an increase of beta-sheet structure in the solid state. The beta-sheet formation of PAIRC peptide may reflect the structural transformation of the serpin reactive center that is relevant to the inhibitor activity. The beta-sheet structure of alpha-synuclein in the solid state may correspond to the amyloid-like aggregates, which are implicated in the pathogenesis of some neurodegenerative diseases.     

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN α-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN α-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN α-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN α-2b remains mostly unchanged at a variety of BSA to IFN α-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN α-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN α-2b secondary structure when BSA is in molar excess.     

Improved estimation of the secondary structures of proteins by vacuum-ultraviolet circular dichroism spectroscopy

The vacuum-ultraviolet circular dichroism (VUVCD) spectra of 16 globular proteins (insulin, lactate dehydrogenase, glucose isomerase, lipase, conalbumin, transferrin, catalase, subtilisin A, alpha-amylase, staphylococcal nuclease, papain, thioredoxin, carbonic anhydrase, elastase, avidin, and xylanase) were successfully measured in aqueous solutions at 25 degrees C from 260 to 160 nm under a high vacuum using a synchrotron-radiation VUVCD spectrophotometer. These proteins exhibited characteristic CD spectra below 190 nm that were related to their different secondary structures, which could not be detected with a conventional CD spectrophotometer. The component spectra of alpha-helices, beta-strands, turns, and unordered structures were obtained by deconvolution analysis of the VUVCD spectra of 31 reference proteins including the 15 proteins reported in our previous paper [Matsuo, K. et al. (2004) J. Biochem. 135, 405-411]. Prediction of the secondary-structure contents using the SELCON3 program was greatly improved, especially for alpha-helices, by extending the short-wavelength limit of CD spectra to 160 nm and by increasing the number of reference proteins. The numbers of alpha-helix and beta-strand segments, which were calculated from the distorted alpha-helix and beta-strand contents, were close to those obtained on X-ray crystallography. These results demonstrate the usefulness of synchrotron-radiation VUVCD spectroscopy for the secondary structure analysis of proteins.     

Calcium-binding properties of cardiac and skeletal troponin C as determined by circular dichroism and ultraviolet difference spectroscopy.

Calcium titration of the conformational change in cardiac and skeletal troponin C (TN-C) was followed by circular dichroism (CD) at pH values in the range from 5.2 to 7.4. Computer analysis was used to resolve the contributions from the different classes of Ca2+ -binding sites. At pH 6.94 in skeletal TN-C, apparent affinity constants for calcium of 1.8 x 10(7) and 4.5 x 10(5) M-1 were determined for the two classes of binding sites. The more sophisticated computer analysis of the data has revealed a substantial CD contribution from the low-affinity sites (approximately 30% of the high affinity contribution at pH 6.94) and suggests that skeletal TN-C with Ca2+ bound at the low-affinity sites is in a different conformation from that when just the high-affinity sites are occupied, in agreement with a recent nuclear magnetic resonance (NMR) study on this system (Seaman, K. B., Hartshorne, D. J. & Bothener-By, A. A. (1977) Biochemistry 16,4039-4046). With the cardiac protein at pH 7.07, an apparent affinity constant for calcium of 2.0 x 10(7) M-1 was calculated while no low-affinity site at this pH was detected by CD. On the other hand, at lower pH values, such as 6.05, a CD contribution from the cardiac low-affinity Ca2+ -binding site is detected with an apparent binding constant of 3.7 +/- 0.7 x 10(4) M-1. At the lower pH values, protonation of a class of carboxyl groups in each protein which possesses a high pKa (6.2-6.3) elicits the conformational change at the high-affinity sites with a corresponding decrease in the overall magnitude of the Ca2+ -evoked changes. The expression of a conformational change upon Ca2+ binding at the level of the low-affinity sites is enchanced by protonation of a class of carboxyls with a pKa of 6.3 in cardiac TN-C and 6.7-6.8 with the skeletal homologue. In both cases, this contribution is reduced upon protonation of carboxyls with pKa less than or equal to 5.5. It was also observed that the low-affinity sites of skeletal TN-C have a much larger role to play in the total conformational change than the low-affinity sites of cardiac TN-C, a finding probably related to the inability of site 1 in the cardiac protein to bind calcium. In the cardiac protein, the Ca2+ -induced tyrosine difference-spectrum maximum is reduced from deltaepsilonM,287nm =330M-1.cm-1 to 20M-1.cm-1 by protonation of a class of groups with a pKa of 6.4, presumably the same carboxyl groups as those invoved in the CD conformational contribution from the high-affinity binding sites. No such effect was observed for the skeletal protein where deltaepsilonM,287nm was constant at 110M-1 .cm-1 over the pH range studied. The dramatic alterations in the tyrosine environment of cardiac TN-C with pH are attributed to either or both of the tyrosines located in the two high-affinity Ca2+ -binding sites (sites 3 and 4)...     

Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction

Heat shock proteins are rapidly synthesized when cells are exposed to stressful agents that cause protein damage. The 70-kDa heat shock induced proteins and their closely related constitutively expressed cognate proteins bind to unfolded and aberrant polypeptides and to hydrophilic peptides. The structural features of the 70-kDa heat shock proteins that confer the ability to associate with diverse polypeptides are unknown. In this study, we have used circular dichroism (CD) spectroscopy and secondary structure prediction to analyze the secondary structure of the mammalian 70-kDa heat shock cognate protein (hsc 70). The far-ultraviolet CD spectrum of hsc 70 indicates a large fraction of alpha-helix in the protein and resembles the spectra one obtains from proteins of the alpha/beta structural class. Analysis of the CD spectra with deconvolution methods yielded estimates of secondary structure content. The results indicate about 40% alpha-helix and 20% aperiodic structure within hsc 70 and between 16-41% beta-sheet and 21-0% beta-turn. The Garnier-Osguthorpe-Robson method of secondary structure prediction was applied to the rat hsc 70 amino acid sequence. The predicted estimates of alpha-helix and aperiodic structure closely matched the values derived from the CD analysis, whereas the predicted estimates of beta-sheet and beta-turn were midway between the CD-derived values. Present evidence suggests that the polypeptide ligand binding domain of the 70-kDa heat shock protein resides within the C-terminal 160 amino acids [Milarski, K. L., & Morimoto, R. I. (1989) J. Cell Biol. 109, 1947-1962].(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the secondary structure of urokinase by the circular dichroism method

The secondary structure of urokinase with molecular weight 33 000 dalton was studied by the circular dichroism method. The secondary structure parameters were calculated based on the protein reference CD spectra resulted in the following secondary structure parameters: approximately 30% aminoacid residues constitute the alpha-helical regions, the same amount forms the beta-structure and an essential fractions contributes the beta-turns. Conformational stability of urokinase to alkaline pH (up to 11.6) and high temperature (up to 80 degrees) in 0.1 M phosphate buffer pH 6.6 was found.     

Using circular dichroism spectra to estimate protein secondary structure

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

The secondary structure of salt-extracted ribosomal proteins from Escherichia coli as studied by circular dichroic spectroscopy

Ribosomal proteins from Escherichia coli MRE600 have been obtained by a new, mild purification procedure. This involves extraction of the subunits with salt followed by chromatographic fractionation in the presence of salt. The use of urea or other denaturing agents and conditions is avoided. A survey of the secondary structure of the 30 S and 50 S proteins, as observed by circular dichroic spectroscopy, is presented. The spectra have been analysed by a new procedure which uses a library of 16 circular dichroic spectra of proteins with a known three-dimensional structure. This method provides a more reliable analysis, especially of the contribution from beta-sheet. The results show that most of the 30 S proteins have a high alpha-helix content, whereas the 50 S proteins are more diverse. The latter group shows a larger contribution from beta-sheet. The data presented here are compared with those already published for a number of proteins which were, with one exception, prepared in the presence of urea. In most cases we find higher alpha-helix and beta-sheet values for the salt-extracted proteins than for the corresponding urea-treated proteins. In those cases, however, where special care was taken to renature the urea-treated proteins agreement is found to within the expected experimental error. The results show that salt-extracted ribosomal proteins have a well-defined secondary structure with a relatively small contribution from unordered structure.