Frank Wien

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.

Calibration and Standardisation of Synchrotron Radiation Circular Dichroism and Conventional Circular Dichroism Spectrophotometers

Synchrotron radiation circular dichroism (SRCD) is an emerging technique in structural biology with particular value in protein secondary structure analyses since it permits the collection of data down to much lower wavelengths than conventional circular dichroism (cCD) instruments. Reference database spectra collected on different SRCD instruments in the future as well as current reference datasets derived from cCD spectra must be compatible. Therefore there is a need for standardization of calibration methods to ensure quality control. In this study, magnitude and optical rotation measurements on four cCD and three SRCD instruments were compared at 192.5, 219, 290 and 490 nm. At high wavelengths, all gave comparable results, however, at the lower wavelengths, some variations were observable. The consequences of these differences on the spectrum, and the calculated secondary structure, of a representative protein (myoglobin) are demonstrated. A method is proposed for standardising spectra obtained on any CD instrument, conventional or synchrotron-based, with respect to existing and future databases.

Biomedical applications of synchrotron radiation circular dichroism spectroscopy: Identification of mutant proteins associated with disease and development of a reference database for fold motifs

Synchrotron radiation circular dichroism (SRCD) spectroscopy is an emerging technique in structural biology with particular value for accurate secondary structure determination, monitoring protein folding and kinetics, and drug discovery. This paper discusses new biomedical applications of SRCD, notably the identification of conformational changes associated with a mutant protein that causes disease, and the development of methods for identification of fold motifs in the context of structural genomics programmes. In addition, it presents for the first time, very low wavelength (below 154 nm) data for a protein in aqueous solution, demonstrating the presence of heretofore-unseen electronic transitions.

VUV irradiation effects on proteins in high-flux synchrotron radiation circular dichroism spectroscopy

Synchrotron radiation circular dichroism (SRCD) spectroscopy is emerging as an important new tool in structural molecular biology. Previously we had shown that in lower-flux SRCD instruments, such as UV1 at ISA and beamline 3.1 at the SRS, vacuum ultraviolet (VUV) radiation damage to proteins was not evident after exposure over a period of hours. No effects were detected in either the protein primary or the secondary structures. However, with the development of high-flux beamlines, such as CD12 at the SRS, this issue has been revisited because of changes observed in the SRCD spectra of consecutive scans of protein samples obtained on this high-flux beamline. Experiments have been designed to distinguish between two different possible mechanisms: (i) photoionization causing free radicals or secondary electrons producing degradation of the protein, and (ii) local heating of the sample resulting in protein denaturation. The latter appears to be the principal source of the signal deterioration.

Anisotropy spectra of amino acids.

Biopolymers such as enzymes and nucleic acids are composed of homochiral monomers; their molecular symmetry is broken. The origin of biomolecular symmetry breaking— a crucial step in the origin of life—remains unknown. Among various random and deterministic hypotheses that have been proposed, one well-known hypothesis is based on a photochemical model by which chiral photons, in the form of circularly polarized (CP) light, induce an enantioenrichment by interacting with racemic organic molecules, a process known as enantioselective photolysis. According to this model, asymmetric photoreactions took place in the extreme vacuum of interstellar space, prior to the delivery of enantioenriched chiral organic molecules to the early Earth. The hypothesis proposes that CP electromagnetic radiation, such as that detected in the Orion molecular cloud, interacts asymmetrically with chiral organic molecules in interstellar ices and with the early precursors of carbonaceous meteorites. Both enantiomers absorb CP photons triggering photolysis, but one enantiomer has a slightly smaller absorption coefficient. This enantiomer is photo-destroyed less rapidly than its optical antipode and it will therefore become enantioenriched. The induced enantiomeric excess (ee) is determined by the extent of reaction x and is function of the anisotropy factor g, defined by De/e, the ratio between the differential extinction coefficient De, and the extinction coefficient e. However, the sign and magnitude of g depend on the wavelength of the CP light. Here we report anisotropy spectra of amino acids yielding g(l) values, which were recorded for solid amorphous films in a wavelength range between 130 and 350 nm. The anisotropy spectra were measured with a new experimental setup at the synchrotron radiation facility ASTRID at Aarhus University (Denmark). The anisotropy spectra obtained for amino acids in the solid phase show well-resolved zero-crossings, extrema, and g values up to 0.024. These data allow: 1) the prediction of the sign of the induced ee, 2) the determination of the kinetics and the ee values of the enantioselective photolysis, and 3) the selection of the wavelength of the CP light best suited for inducing enantioenrichment. The enantioselective photolysis of a racemic mixture by CP light is an asymmetric transformation that can be represented by two competitive pseudo-first-order reactions with unequal rate constants, kR and kS, for the R and S enantiomer, respectively. The rate constants are proportional to the molar absorption coefficients (eR and eS, respectively), and the efficiency of the enantioselective photolysis depends on the difference between kR and kS or, in this case as Kuhn already outlined, on the anisotropy factor g [Eq. (1)]. 6] More recently it has been shown by Nakamura et al. that Equation (1) is valid even for non-firstorder kinetics.

Reversible assembly of a drug peptide into amyloid fibrils: a dynamic circular dichroism study

The common view on the amyloid fibril formation is that it is a multistep process that involves many oligomeric intermediate species, which leads to a high degree of polymorphism. This view derives from numerous kinetic studies whose vast majority was carried out with amyloid β fragments or other pathological amyloidogenic sequences. Yet, it is not clear whether the mechanisms inferred from these studies are universal and also apply to functional amyloids, in particular to peptide hormones which form reversible amyloid structures. In the present work, we study the self-assembly properties of atosiban, a nonapeptide drug, whose sequence is very close to those of the oxytocin and vasopressin hormones. We show that this very soluble peptide consistently self-assembles into 7 nm wide amyloid fibrils above a critical aggregation concentration (2-10 w/w % depending on the buffer conditions). The peptide system is characterized in details, from the monomeric to the assembled form, with osmotic concentration measurements, transmission electron microscopy, small-angle X-ray scattering, infrared and fluorescence spectroscopy, and circular dichroism (CD). We have followed in situ the fibril assembly with fluorescence and synchrotron radiation CD and noticed that the peptide undergoes conformational changes during the process. However, several lines of evidence point toward the association of monomers and dimers into fibrils without passing through oligomeric intermediate species contrary to what is usually reported for pathogenic amyloids. The native β-hairpin conformation of the monomer could explain the straightforward assembly. The tyrosine stacking is also shown to play an important role.