Synchrotron Radiation Circular Dichroism Data Research Articles

Structural features and stability of apo- and holo-forms of a simple iron-sulfur protein.

Iron-sulfur centers are widespread in living organisms, mostly performing electron transfer functions, either in electron transfer chains or as part of multi-enzymatic complexes, while being also present in enzyme active sites, handling substrate catalysis. Rubredoxin is the simplest iron-sulfur containing protein constituted by a single polypeptide chain of 50 to 60 amino acids, of which four cysteine residues are responsible for metal binding in a tetrahedral coordination sphere. In this manuscript we explore the structure and stability of both apo- and holo-forms of a Rubredoxin from Marinobacter hydrocarbonoclasticus using Synchrotron Radiation Circular Dichroism (SRCD) in combination with other biochemical and spectroscopic techniques. The results are consistent with a holo-protein form containing a monomeric iron center with UV-visible maxima at 760, 578, 494, 386, 356and 279nm, an intense EPR resonance with a g value around 4.3 and Mössbauer spectroscopy parameters of δ equal to 0.69mm/s and ΔEQ equal to 3.25mm/s, for the ferrous reconstituted state. SRCD data, obtained for the first time for the apo-form, show a quite defined structure with ∆ε maximum at 191nm and minima at 203 and 231nm. Most significantly, the presence of isosbestic points at 189 and 228nm made the interconversion between the two stable apo- and holo-form solution structures clear. SRCD temperature dependence data shows that for both forms the denaturation process proceeds through an intermediate species.

Dps-DNA interaction in Marinobacter hydrocarbonoclasticus protein: effect of a single-charge alteration.

DNA-binding proteins from starved cells (Dps) are members of the ferritin family of proteins found in prokaryotes, with hollow rounded cube-like structures, composed of 12 equal subunits. These protein nanocages are bifunctional enzymes that protect the cell from the harmful reaction of iron and peroxide (Fenton reaction), thus preventing DNA damage by oxidative stress. Ferrous ions are oxidized at specific iron-binding sites in the presence of the oxidant and stored in its cavity that can accommodate up to ca. 500 iron atoms. DNA-binding properties of Dps are associated with the N-terminal, positive charge rich, extensions that can promote DNA binding and condensation, apparently by a cooperative binding mechanism. Here, we describe the binding and protection activities of Marinobacter hydrocarbonoclasticus Dps using Electrophoretic Mobility Shift Essays (EMSA), and synchrotron radiation circular dichroism (SRCD) spectroscopy. While no DNA condensation was observed in the tested conditions, it was possible to determine a Dps-DNA complex formation with an apparent dissociation constant of 6.0± 1.0µM and a Hill coefficient of 1.2 ± 0.1. This interaction is suppressed by the inclusion of a single negative charge in the N-terminal region by point mutation. In Dps proteins containing a ferric mineral core (above 96 Fe/protein), DNA binding was impaired. SRCD data clearly showed that no significant modification existed either in secondary structure or protein stability of WT, Q14E variant and core containing proteins. It was, however, interesting to note that, in our experimental conditions, thermal denaturation induced protein aggregation that caused artifacts in thermal denaturation curves, which were dependent on radiation flux and vertical arrangement of the CD cell.

Circular-Dichroism and Synchrotron-Radiation Circular-Dichroism Spectroscopy as Tools to Monitor Protein Structure in a Lipid Environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190nm and producing information that is unobtainable by conventional CD instruments. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

The physicochemical properties of membranes correlate with the NADPH oxidase activity

BackgroundPhagocytes kill ingested microbes by exposure to high concentrations of toxic reactive species generated by NADPH-oxidases. This membrane-bound electron-transferring enzyme is tightly regulated by cellular signaling cascades. So far, molecular and biophysical studies of the NADPH-oxidase were performed over limited temperature ranges, which weaken our understanding of immune response or inflammatory events. In this work, we have inspected the influence of temperature and lipid membrane properties on the NADPH-oxidase activity using a system free of cell complexity. MethodsWe have extended the experimental conditions of the accepted model for NADPH-oxidase activity, the so-called cell-free assay, to a large temperature range (10–40°C) using different membrane compositions (subcellular compartments or liposomes). ResultsA remarkable increase of superoxide production rate was observed with rising temperature. Synchrotron radiation circular dichroism data showed that this is not correlated with protein secondary structure changes. When lipid bilayers are in fluid phase, Arrhenius plots of the oxidase activity showed linear relationships with small activation energy (Ea), while when in solid phase, high Ea was found. The sterol content modulates kinetic and thermodynamic parameters. ConclusionHigh temperature promotes the rate of superoxide production. The key element of this enhancement is related to membrane properties such as thickness and viscosity and not to protein structural changes. Membrane viscosity that can be driven by sterols is a paramount parameter of Ea of NADPH oxidase activity. The membrane bilayer state modulated by its sterol content may be considered locally as an enzyme regulator.This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.

Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst).

Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst) were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD) and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM) and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

On the wavelength dependence of UV induced thymine photolesions: a synchrotron radiation circular dichroism study.

Solar mutagenesis via the formation of thymine dimer photoproducts is a primary cause of skin cancer. The aim of this study is to provide a direct method for following the development of photolesions in thymine single strands and to determine how the formation of these photoproducts depends on the excitation wavelength in the ultraviolet (UV) between 210 nm and 325 nm. Experiments were performed both with a 20 Hz pulsed, intense, tunable laser as well as UV lamps (at 254 nm and 302 nm), but we find that only the dose matters at these wavelengths for the yield of photoproducts. Hence in both cases the lesion process is due to one-photon absorption. The formation and yields of the photoproducts as the irradiation dose is increased is followed through measurement of synchrotron radiation circular dichroism (SRCD) spectra. A principal component analysis (PCA) of the SRCD data yields CD signatures for each of the resulting photoproducts and reveals a strong irradiation wavelength dependence upon which products are formed; cyclobutane pyrimidine dimers (CPDs) are formed primarily at higher irradiation wavelengths (from 250 to 300 nm); the 6,4 pyrimidine-pyrimidone photoadduct (64PP) is formed in the range 210 to 285 nm, with a higher rate of formation in the lower part of that range, while in the very lowest irradiation wavelength range (210 to 240 nm) we find thymidine monophosphate (dTMP), which indicates cleavage of the DNA backbone. Our work demonstrates the strength of SRCD spectroscopy compared to ordinary absorption spectroscopy, as the latter is not sufficient to obtain fingerprints of the thymine photoproducts.

Molecular and immunological characterization of wheat Serpin (Tri a 33)

Several wheat proteins are responsible for food and respiratory allergies. Due to their large polymorphism, the allergenic potential of a number of them has not yet been precisely established. The aim of this work was to perform a thorough assessment of serpin (Tri a 33) allergenicity. Recombinant wheat Serpin-Z2B isoform (rSerpin-Z2B) was expressed in Escherichia coli. Synchrotron radiation circular dichroism data indicated that the recombinant serpin contains slightly more β-strands than α-helix structures. IgE reactivity of sera from 103 patients with food allergy and 29 patients with Baker's asthma was evaluated using ELISA, a model of basophil activation and linear epitope mapping (Pepscan). Twenty percent of patients with food allergy to wheat and 31% of those with Baker's asthma displayed rSerpin-Z2B-specific IgE in ELISA. The protein was able to induce IgE-dependent basophil degranulation. The Pepscan experiment identified four regions involved in IgE binding to serpin. Heating the protein induced its irreversible denaturation and impaired IgE binding, revealing the predominance of conformational epitopes. This study confirms wheat serpin allergenicity and shows that recombinant serpin may be a marker of a broad spectrum of sensitization to wheat proteins.

JCAMP-DX for circular dichroism spectra and metadata (IUPAC Recommendations 2012)

Circular dichroism (CD) spectroscopy is a widely used technique for the characterisation of proteins. A number of CD instruments are currently on the market, and there are more than a dozen synchrotron radiation circular dichroism (SRCD) beamlines in operation worldwide. All produce different output formats and contents. In order for users of CD and SRCD data to be able simply to compare and contrast data and the associated recorded or unrecorded metadata, it is essential to have a common data format. For this reason, the JCAMP-DX-CD format for CD spectroscopy has been developed, based on extensive consultations with users and senior representatives of all the instrument manufacturers and beamlines, and under the auspices of IUPAC, based on the Joint Committee on Atomic and Physical Data Exchange protocols. The availability of a common format is also important for deposition to, and access from, the Protein Circular Dichroism Data Bank, the public repository for CD and SRCD data and metadata. The JCAMP-DX-CD format can be read by standard JCAMP programs such as JSpecView. We have also created a series of parsers, available at the DichroJCAMP web site (http://valispec.cryst.bbk.ac.uk/formatConverter/dichroJCAMPDX-CD.html), which will enable the conversion between instrument and beamline formats and the JCAMP-DX-CD format.

Predictions of Protein Circular Dichroism Calculated by the Dipole Interaction Model and Compared to Synchrotron Radiation Circular Dichroism Experiments

The determination of accurate secondary, tertiary and quaternary structures of macromolecules is vital for the study of intra- and interbimolecular interactions important for drug discovery, probing enzyme mechanisms and other functional protein complexes. Despite the recent advances in protein structural determination methods like X-ray crystallography, there is still a need to evaluate the accuracy of the model structures used in macromolecular interaction prediction. This is especially important where accurate three dimensional structures of the proteins are absent. Synchrotron radiation circular dichroism (SRCD) is an emerging technique sensitive not only to secondary structure but also teriary and quatertary structure. Theoretical calculations of circular dichroism (CD) using the dipole interaction model have successfully predicted CD for a variety of peptides and monomeric proteins. Herein, the dipole interaction model predicts CD for proteins in monomeric, dimmeric or tetrameric forms and is compared to experimental SRCD spectra. This is an attempt to evaluate the homology models. Preliminary results show comparable CD spectra between the dipole interaction model predictions and SRCD data for small proteins like myoglobin.View Large Image | View Hi-Res Image | Download PowerPoint Slide

3‐ Instead of 4‐Helix Formation in a De Novo Designed Protein in Solution Revealed by Small‐Angle X‐ray Scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability.

The effects of lipids on the structure of the eukaryotic cytolysin equinatoxin II: A synchrotron radiation circular dichroism spectroscopic study

Synchrotron radiation circular dichroism (SRCD) spectroscopy studies of the eukaryotic pore-forming protein equinatoxin II (EqtII) were carried out in solution and in the presence of micelles or small unilamellar vesicles (SUV) of different lipid composition. The SRCD structural data was correlated with calcein leakage from SUV and with partitioning of EqtII to liposomes, and micelles, according to haemolysis assays. The structure of EqtII in water and dodecylphosphocholine micelles as determined by SRCD was similar to the values calculated from crystal and solution structures of the protein, and no changes were observed with the addition of sphingomyelin (SM). SM is required to trigger pore formation in biological and model membranes, but our results suggest that SM alone is not sufficient to trigger dissociation of the N-terminal helix and further structural rearrangements required to produce a pore. Significant changes in conformation of EqtII were detected with unsaturated phospholipid (DOPC) vesicles when SM was added, but not with saturated phospholipids (DMPC), which suggests that not only is membrane curvature important, but also the fluidity of the bilayer. The SRCD data indicated that the EqtII structure in the presence of DOPC:SM SUV represents the ‘bound’ state and the ‘free’ state is represented by spectra for DOPC or DOPC:Chol vesicles, which correlates with the high lytic activity for SUV of DOPC:SM. The SRCD results provide insight into the lipid requirements for structural rearrangements associated with EqtII toxicity and lysis.

Combining sequence-based prediction methods and circular dichroism and infrared spectroscopic data to improve protein secondary structure determinations

BackgroundA number of sequence-based methods exist for protein secondary structure prediction. Protein secondary structures can also be determined experimentally from circular dichroism, and infrared spectroscopic data using empirical analysis methods. It has been proposed that comparable accuracy can be obtained from sequence-based predictions as from these biophysical measurements. Here we have examined the secondary structure determination accuracies of sequence prediction methods with the empirically determined values from the spectroscopic data on datasets of proteins for which both crystal structures and spectroscopic data are available.ResultsIn this study we show that the sequence prediction methods have accuracies nearly comparable to those of spectroscopic methods. However, we also demonstrate that combining the spectroscopic and sequences techniques produces significant overall improvements in secondary structure determinations. In addition, combining the extra information content available from synchrotron radiation circular dichroism data with sequence methods also shows improvements.ConclusionCombining sequence prediction with experimentally determined spectroscopic methods for protein secondary structure content significantly enhances the accuracy of the overall results obtained.