Circular dichroism and DNA secondary structure.

The change in average rotation of the DNA helix has been determined for the transfer from 0.05 M NaCl to 3.0 M CsCl, 6.2 M LiCl and 5.4 M NH4Cl. This work, combined with data at lower salt from other laboratories, allows us to relate the intensity of the CD of DNA at 275 nm directly to the change in the number of base pairs per turn. The change in secondary structure for the transfer of DNA from 0.05 M NaCl (where it is presumably in the B-form) to high salt (where the characteristic CD has been interpreted as corresponding to C-form geometry) is found to be -0.22 (+/- 0.02) base pairs per turn. In the case of mononucleosomes, where the CD indicates the "C-form", the change in secondary structure (including temperature effects) would add -0.31 (+/- 0.03) turns about the histone core to the -1.25 turns estimated from work on SV40 chromatin. Accurate winding angles and molar extinction coefficients were determined for ethidium.     

Circular dichroism spectroscopy has intrinsic limitations for protein secondary structure analysis

Secondary structure content (SSC) cannot be calculated accurately from circular dichroism (CD) spectra for the majority of proteins whose three-dimensional structures have been solved. “Reliable” SSC that is significantly different from random SSC can be calculated from CD spectra only for all-α proteins and all-β proteins with canonical β-strand geometry.     

Circular dichroism and the secondary structure of the ROF2 protein from Arabidopsis thaliana

The protein ROF2 from the plant Arabidopsis thaliana acts as a heat stress modulator, being involved in the long-term acquired thermotolerance of the plant. Here we investigate the relationship between the biological function and the structure of ROF2, inferred by circular dichroism (CD) spectroscopy. The far-UV CD spectra, analyzed with the CDPro and DICHROWEB program packages, yield the percentages of α-helices, β-sheets, unordered regions, turns and poly(Pro)II-helices in the secondary structure of ROF2. According to the analysis, the percentages of the structural elements of ROF2 are about 40% for β-sheets, 30% for unordered regions, 17% for turns, 10% for poly(Pro)II-helices and 3% for α-helices. The near-UV CD spectra suggest that ROF2 proteins can associate, forming super-secondary structures. Our CD experiments performed at temperatures between 5 °C and 97 °C indicate that the thermal denaturation of ROF2 caused by a raise in temperature up to 55 °C is followed by a thermal refolding of the protein as the temperature is raised further. The new secondary structure, acquired around 65 °C, remains stable up to 97 °C. The structural stability of ROF2 at high temperatures might play an important role in the experimentally observed thermotolerance of Arabidopsis thaliana.     

Concentration-independent estimation of protein secondary structure by circular dichroism: a comparison of methods

Estimation of a protein's secondary structure from its circular dichroism spectrum usually requires accurate knowledge of the concentration and pathlength of the sample. Two recently described methods avoid this problem by analysis of g-factor spectra (McPhie, Anal. Biochem. 293, 109-119) or scaling of relative intensities (Raussens et al., Anal. Biochem. 319, 114-121). Application of the two methods to the same samples shows that they can have similar efficacies. Calculation with the latter method is more rapid, but the performance of the former is maintained over reduced wavelength ranges.     

Circular dichroism studies on proteins in films and in solution: estimation of secondary structure by g-factor analysis

Estimates of the secondary structure of a protein in solution are made by mathematical analyses of its circular dichroism (CD) spectrum below 240 nm. All current procedures require accurate determination of the concentration of the protein sample. Insoluble proteins, such as prions or amyloid, are examined as thin films or gels, but concentrations cannot be precisely defined. The ratio of a sample's CD and absorbance signals is the g-factor, an intensive property. The g-factor spectra of 19 soluble, unconjugated proteins of known structures were measured and used to derive basis spectra, characteristic of the four major structural elements, helix, sheet, turn, and remainder. Using these, the g-factor spectra of other unconjugated proteins, measured in solution or as films, can be analyzed by linear regression to give good estimates of their secondary structures when protein concentration cannot be determined.     

Circular dichroism analysis of the secondary structure of Z protein and its complexes with bilirubin and other organic anions.

Circular dichroism (CD) methods were employed to study the conformation of Z protein and characterize its complexes with bilirubin and other organic anions. Z protein-bilirubin complexes exhibited a spectrum with overlapping ellipticity bands of opposite sign in the bilirubin absorption region. These results were compared with those obtained with ligandin, the other major organic anion binding protein of liver. Secondary structural differences between the two proteins were easily demonstrated since ligandin is predominantly an alpha-helical protein and Z features mainly beta-structure. Furthermore, the optical activity pattern generated by bilirubin binding to Z was virtually a mirror image of that of the ligandin bilirubin system. CD experiments were designed to study the direct transfer of bilirubin between Z protein and ligandin, and it was shown that both proteins have almost equal affinities for bilirubin. The bilirubin on Z was readily displaced by oleic acid and displaced to a lesser extent by sulfobromophthalein,     

Synchrotron radiation circular dichroism spectroscopy of proteins: secondary structure, fold recognition and structural genomics

Recent developments in instrumentation and bioinformatics show that the technique of synchrotron radiation circular dichroism spectroscopy can provide novel information on protein secondary structures and folding motifs, and has the potential to play an important role in structural genomics studies, both as a means of target selection and as a high-throughput, low-sample-requiring screening method. This is possible because of the additional information content in the low-vacuum ultraviolet wavelength data obtainable with intense synchrotron radiation light sources, compared with that present in spectra from conventional lab-based circular dichroism instruments.     

Terahertz Radiation at ANKA, the New Synchrotron Light Source in Karlsruhe

ANKA is a new synchrotron light source atthe Karlsruhe Research Center in southwestGermany. The acronym stands for Ångstrøm Source Karlsruhe.The ANKA-IR beamline provides a highbrilliance infrared beam through the near,mid and far-infrared range. Thefar-infrared range is of particularinterest, since at frequencies lower thanaround 200 cm^-1 (6 THz) synchrotronlight begins to outperform conventionalthermal sources in terms of total intensityas well as brilliance. The extraction ofthe entire flux is a challenge in the THzrange, since the natural verticaldivergence of synchrotron radiationincreases with wavelength and the openingangle for collection is limited by designconstraints. At ANKA-IR, this problem issolved by the collection of radiationemitted from a bending magnet edge source,which has a much smaller verticaldivergence than conventional synchrotronradiation emitted from the constantmagnetic field region within the dipolemagnet. Edge radiation at ANKA permits theextraction of the entire infrared flux downto around 100 cm^-1 (3 THz) while withconventional synchrotron radiation thiswould only be the case for frequencies downto 2500 cm^-1. ANKA-IR provides usableintensity down to 4 cm^-1 (120 GHz).     

BMIT facility at the Canadian Light Source: Advances in X-ray phase-sensitive imaging

The BioMedical Imaging and Therapy (BMIT) facility [1,2] located at the Canadian Light Source, provides synchrotron-specific imaging and radiation therapy capabilities. There are two separate beamlines used for experiments: the bending magnet (05B1-1) and the insertion device (05ID-2) beamline.The bending magnet beamline provides access to monochromatic beam spanning a spectral range of 15–40 keV, and the beam is 240 mm wide in the POE-2 experimental hutch. Users can also perform experiments with polychromatic (pink) beam.The insertion device beamline was officially opened for general user program in 2015. The source for the ID beamline is a multi-pole, superconducting 4.3 T wiggler. The high field gives a critical energy over 20 keV. The optics hutches prepare a beam that is 220 mm wide in the last experimental hutch SOE-1. The monochromatic spectral range spans 25–150+ keV. Several different X-ray detectors are available for both beamlines, with resolutions ranging from 2 μm to 200 μm.BMIT provides a number of imaging techniques including standard absorption X-ray imaging, K-edge subtraction imaging (KES), in-line phase contrast imaging (also known as propagation based imaging, PBI) and Diffraction Enhanced Imaging/Analyzer Based Imaging (DEI/ABI), all in either projection or CT mode. PBI and DEI/ABI are particularly important tools for BMIT users since these techniques enable visualization of soft tissue and allow for low dose imaging.     

dam methylase from E. coli. Circular dichroism investigations of the secondary structure and influence of S-adenosylmethionine

The enzyme dam methylase which recognizes and methylates the adenine in the palindromic sequence GATC in DNA was isolated and the secondary structure was determined by CD spectroscopy and various predicting methods from the amino acid sequence. The interaction of dam methylase with S-adenosylmethionine was studied by CD spectroscopy indicating a decrease of the percentage of alpha-helix as the amount of S-adenosylmethionine bound to the enzyme was increased.     

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 and laser Raman spectroscopic analysis of the secondary structure ofCerebratulus lacteus toxin B-IV

The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation.     

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.     

The optimization of protein secondary structure determination with infrared and circular dichroism spectra.

We have used the circular dichroism and infrared spectra of a specially designed 50 protein database [Oberg, K.A., Ruysschaert, J.M. & Goormaghtigh, E. (2003) Protein Sci. 12, 2015-2031] in order to optimize the accuracy of spectroscopic protein secondary structure determination using multivariate statistical analysis methods. The results demonstrate that when the proteins are carefully selected for the diversity in their structure, no smaller subset of the database contains the necessary information to describe the entire set. One conclusion of the paper is therefore that large protein databases, observing stringent selection criteria, are necessary for the prediction of unknown proteins. A second important conclusion is that only the comparison of analyses run on circular dichroism and infrared spectra independently is able to identify failed solutions in the absence of known structure. Interestingly, it was also found in the course of this study that the amide II band has high information content and could be used alone for secondary structure prediction in place of amide I.     

SOMCD: method for evaluating protein secondary structure from UV circular dichroism spectra

This article presents SOMCD, an improved method for the evaluation of protein secondary structure from circular dichroism spectra, based on Kohonen's self-organizing maps (SOM). Protein circular dichroism (CD) spectra are used to train a SOM, which arranges the spectra on a two-dimensional map. Location in the map reflects the secondary structure composition of a protein. With SOMCD, the prediction of beta-turn has been included. The number of spectra in the training set has been increased, and it now includes 39 protein spectra and 6 reference spectra. Finally, SOM parameters have been chosen to minimize distortion and make the network produce clusters with known properties. Estimation results show improvements compared with the previous version, K2D, which, in addition, estimated only three secondary structure components; the accuracy of the method is more uniform over the different secondary structures.