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.     

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.     

Structural conformation of spidroin in solution: a synchrotron radiation circular dichroism study

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.     

Vacuum-ultraviolet circular dichroism study of saccharides by synchrotron radiation spectrophotometry

Vacuum-ultraviolet circular dichroism (VUVCD) spectra of five monosaccharides (D-glucose, D-mannose, D-galactose, D-xylose, and D-lyxose) and five disaccharides (maltose, isomaltose, cellobiose, gentiobiose, and lactose) were measured to 160 nm using a synchrotron-radiation VUVCD spectrophotometer in aqueous solution under high vacuum at 25 degrees C. Most of the saccharides show a positive peak with some shoulders at around 170 nm, except for D-galactose and lactose, which show two distinct negative peaks at around 165 and 177 nm. These spectra are influenced by such structural factors as alpha and beta anomers at C-1, axial and equatorial hydroxyl groups at C-2 and C-4, trans (T) and gauche (G) conformations of the hydroxymethyl group at C-5, and the type of glycosidic linkage. Deconvolution of the VUVCD spectra of D-glucose, D-mannose, and D-galactose into six independent Gaussian components for alpha-GG, alpha-GT, alpha-TG, beta-GG, beta-GT, and beta-TG conformations suggests that the alpha anomer has red-shifted spectra relative to the beta anomer, and that GG and GT conformations have positive and negative circular dichroism signs, respectively, while the sign for TG conformation is anomer dependent. These speculations from the deconvolution analyses are also supported by the VUVCD spectra of disaccharides. These results give new insight into the equilibrium conformations of saccharides, demonstrating the usefulness of synchrotron-radiation VUVCD spectroscopy.     

Structure and activity of the N-terminal region of the eukaryotic cytolysin equinatoxin II

The actinoporins are a family of proteins from sea anemones that lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being resistant to these toxins. Pore formation by the actinoporin equinatoxin II (EqTII) proceeds by membrane binding via a surface rich in aromatic residues, followed by translocation of the N-terminal region to the membrane and, finally, across the bilayer to form a functional pore. A key feature of this mechanism is the ability of the N-terminal region to form a stable, bilayer-spanning helix in the membrane, which in turn requires dissociation of the N-terminus from the bulk of the protein and significant extension of the N-terminal helix of native EqTII. In this study the structures of three peptides corresponding to residues 11-29, 11-32, and 1-32, respectively, of EqTII have been investigated by high-resolution nuclear magnetic resonance and Fourier transform infrared spectroscopy. The 32-residue peptide lacks ordered secondary structure in water, but residues 6-28 form a helix in dodecylphosphocholine micelles. Although this helix is long enough to span a bilayer membrane, this peptide and the shorter analogues display limited permeabilizing activity in large unilamellar vesicles and very weak hemolytic activity in human red blood cells. Thus, while the N-terminal region has the structural features required for this unusual mechanism of pore formation, the lack of activity of the isolated N-terminus shows that the bulk of the protein is essential for efficient pore formation by facilitating initial membrane binding, interacting with sphingomyelin, or stabilizing the oligomeric pore.     

Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy

Circular dichroism (CD) spectroscopy beamlines at synchrotrons produce dramatically higher light flux than conventional CD instruments. This property of synchrotron radiation circular dichroism (SRCD) results in improved signal-to-noise ratios and allows data collection to lower wavelengths, characteristics that have led to the development of novel SRCD applications. Here we describe the use of SRCD to study protein complex formation, specifically evaluating the complex formed between carboxypeptidase A and its protein inhibitor latexin. Crystal structure analyses of this complex and the individual proteins reveal only minor changes in secondary structure of either protein upon complex formation (i.e., it involves only rigid body interactions). Conventional CD spectroscopy reports on changes in secondary structure and would therefore not be expected to be sensitive to such interactions. However, in this study we have shown that SRCD can identify differences in the vacuum ultraviolet CD spectra that are significant and attributable to complex formation.     

New synchrotron radiation circular dichroism end-station on DISCO beamline at SOLEIL synchrotron for biomolecular analysis

The novel Synchrotron Radiation Circular Dichroism (SRCD) technique is becoming a new tool of investigation for the molecular structures of biomolecules, like proteins, carbohydrates or others bio-materials. Here, we describe the characteristics of a new experimental end-station for circular dichroism studies, in construction on DISCO beamline at SOLEIL synchrotron (Saint-Aubin, France). This experimental end-station will be an open facility for the community of researchers in structural biology. In order to show the kind of information accessible with this type of technique, we give an example: the conformational study of the galactose mutarotase from Escherichia coli, an enzyme involved in the galactose metabolism. This study was made using an operational SRCD station available at SRS (Daresbury Laboratory, UK).     

Interaction of the pore-forming protein equinatoxin II with model lipid membranes: A calorimetric and spectroscopic study.

The interactions of equinatoxin II (EqTxII) with zwitterionic (DPPC) and anionic (DPPG) phospholipids and an equimolar mixture of the two phospholipids (DPPC/DPPG) have been investigated by differential scanning calorimetry (DSC), CD-spectropolarimetry, intrinsic emission fluorescence spectroscopy, and ultrasonic velocimetry. EqTxII binds to small unilamellar vesicles formed from negatively charged DPPG lipids, causing a marked reduction in the cooperativity and enthalpy of their gel/liquid-crystalline phase transition. This transition is completely abolished at a lipid-to-protein ratio, L/P, of 10. For the mixed DPPC/DPPG vesicles, a 2-fold greater lipid-to-protein ratio (L/P = 20) is required to abolish the phase transition, which corresponds to the same negative charge (-10) of lipid molecules per EqTxII molecule. The disappearance of the phase transition of the lipids apparently corresponds to the precipitation of the lipid-protein complex, as suggested by our sound velocity measurements. Based on the far-UV CD spectra, EqTxII undergoes two structural transitions in the presence of negatively charged vesicles (DPPG). The first transition coincides with the gel/liquid-crystalline phase transition of the lipids, which suggests that the liquid-crystalline form of negatively charged lipids triggers structural changes in EqTxII. The second transition involves the formation of alpha-helical structure. Based on these observations, we propose that, in addition to electrostatic interactions, hydrophobic interactions play an important role in EqTxII-membrane association.     

Influence of divalent cations on the conformation of phosphorothioate oligodeoxynucleotides: a circular dichroism study

Phosphorothioate oligodeoxynucleotides (ODNs) have been extensively investigated in vivo and in vitro for antisense control of gene expression. It has been shown that cellular uptake of phosphorothioate ODNs in some in vitro cell systems increases in the presence of divalent cations. In this work, we analyze the conformation of phosphorothioate ODNs and specific changes induced in it by various divalent cations using circular dichroism (CD) spectroscopy. CD data were obtained with several phosphorothioate ODNs in the absence and presence of the divalent cations Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺ and Mn²⁺. All CD spectra indicated stable conformations of the ODNs in solution. The spectra were strongly dependent on ODN sequence and composition. Some ODNs such as T₂₃ and another with ‘random’ distribution of bases showed CD spectra characteristic of B-form DNA. Other ODNs which had at least three consecutive guanines in their sequences exhibited spectra characteristic of parallel G-tetraplexes. CD spectra of antisense ODNs exhibited specific responses to divalent cations. Changes in the conformation were not simply due to ionic strength effects. Mn²⁺ diminished secondary structure in some ODNs. Group II divalent ions stabilized the parallel G-tetraplexes, and Mg²⁺ generally had the weakest stabilizing efficiency. Each sequence/ion combination had a specific response so these effects cannot be generalized. These sequence-dependent, divalent ion-sensitive, and structurally unique solution conformations may be related to ion-mediated ODN uptake.     

The effect of nonenzymatic glycation on the stability and conformation of two deoxyoligonucleotide duplexes: a spectroscopic analysis by circular dichroism

Advanced glycation end products (AGEs) play a significant role in the pathophysiology of diabetes leading to such conditions as atherosclerosis, cataract formation, and renal dysfunction. While the formation of nucleoside AGEs was previously demonstrated, no extensive studies have been performed to assess the effect of AGEs on DNA structure and folding. The objective of this study was to investigate the nonenzymatic glycation of two DNA oligonucleotide duplexes with one duplex consisting of deoxy-poly(A)15 and deoxy-poly(T)15 and the other consisting of deoxy-poly(GA)15 and deoxy-poly(CT)15. With D-glucose, D-galactose, D/L-glyceraldehyde, and D-glucosamine serving as the model glycating carbohydrates, D-glucosamine was found to exhibit the greatest effect on the stability and structure of the oligonucleotide duplexes, a finding that was confirmed by circular dichroism. The nonenzymatic glycation of deoxy-poly(AT) by D-glucosamine destabilized the deoxy-poly(AT) structure and changed its conformation from A form to X form. D-glucosamine also altered the conformation of deoxy-poly(GA)15 and deoxy-poly(CT)15 from A form to B form. Capillary electrophoresis and ultraviolet and fluorescence spectroscopy revealed that, of the various purines and pyrimidines, 2'-deoxyguanosine and guanine were most reactive with D-glucosamine. The nonenzymatic modification of nucleic acids warrants further investigation because this phenomenon may occur in vivo, altering DNA structure and/or function.     

A study of tubulin dimer conformation by near-UV circular dichroism

The near-UV circular dichroism properties of tubulin dimer have been measured for different preparative methods. Tubulin dimer was obtained from assembly compenent microtubule protein by gel filtration, or phosphocellulose ion-exchange chromatography in the presence of magnesium. Tubulin dimer prepared by the protocol of Weisenberg R.C. and Timasheff, S.N. (1970) Biochemistry 9, 4110–4116, was found to be markedly different due to some apparently irreversible change in conformation. We conclude that the removal of microtubule-associated proteins by phosphocellulose ion-exchange chromatography in the presence of magnesium can be performed without affecting the conformation of native tubulin dimer as judged by near-UV circular dichroism.     

Ca(2+)-induced changes of surfactin conformation: a FTIR and circular dichroism study

Previous NMR studies on surfactin proposed two gamma or beta-turn-containing conformers while recent CD studies described beta-sheets and alpha-helices in surfactin. Since these data were not obtained in the same conditions, the conformation of surfactin was reinvestigated by FTIR spectroscopy, a diagnostic method for beta-sheets. In trifluoroethanol, the FTIR spectra of surfactin and its diester are compatible with gamma and/or beta-turn(s) and the differences in their CD spectra show the importance of the Glu(1) and Asp(5) COOH groups in stabilizing the lipopeptide conformation. The calcium-induced spectral changes of both lipopeptides suggest a first binding of the divalent ions to the surfactin COOH groups (until calcium-lipopeptide mole ratio reached 1) followed by bulk conformational changes (at higher mole ratios). In Tris buffer at pH 8.5, the FTIR amide I band shape, without the typical 1610-1628 and 1675-1695 cm(-1) bands, ascertains the absence of beta-sheets.     

Characterization of dry globular proteins and protein fibrils by synchrotron radiation vacuum UV circular dichroism

Circular dichroism using synchrotron radiation (SRCD) can extend the spectral range down to approximately 130 nm for dry proteins, potentially providing new structural information. Using a selection of dried model proteins, including alpha-helical, beta-sheet, and mixed-structure proteins, we observe a low-wavelength band in the range 130-160 nm, whose intensity and peak position is sensitive to the secondary structure of the protein and may also reflect changes in super-secondary structure. This band has previously been observed for peptides but not for globular proteins, and is compatible with previously published theoretical calculations related to pi-orbital transitions. We also show that drying does not lead to large changes in the secondary structure and does not induce orientational artifacts. In combination with principal component analysis, our SRCD data allow us to distinguish between two different types of protein fibrils, highlighting that bona fide fibrils formed by lysozyme are structurally more similar to the nonclassical fibrillar aggregates formed by the SerADan peptide than with the amyloid formed by alpha-synuclein. Thus, despite the lack of direct structural conclusions, a comprehensive SRCD-based database of dried protein spectra may provide a useful method to differentiate between various types of supersecondary structure and aggregated protein species.     

Measuring circular dichroism in a capillary cell using the b23 synchrotron radiation CD beamline at diamond light source

Synchrotron radiation circular dichroism (SRCD) is a well-established method in structural biology. The first UV-VIS beamline dedicated to circular dichroism at Diamond Light Source, a third generation synchrotron facility in South Oxfordshire, has recently become operational and it is now available for the user community. Herein we present an important application of SRCD: the CD measurement of protein solutions in fused silica rectangular capillary cells. This was achieved without the use of any lens between the photoelastic modulator and the photomultiplier tube detectors by exploiting the high photon flux of the collimated beam that can be as little as half a millimeter squared. Measures to minimize or eliminate vacuum-UV protein denaturation effects are discussed. The CD spectra measured in capillaries is a proof of principle to address CD measurements in microdevice systems using the new B23 SRCD beamline.     

A study of the conformation of native and immobilized human somatotropin using circular dichroism

The conformation of human growth hormone (hGH) as monomer and aggregate as well as of those immobilized in the soluble polysaccharide matrix was investigated. The immobilization resulted in ten per cent decrease of the amount of alpha-helix and in the conformational mobility change of aromatic amino acid side groups. It did not, however, influence the biological activity of the hormone, thus suggesting that the above conformational variations did not affect functionally important portions of the molecule.     

Effect of conformation and configuration of arabinose residues on the circular dichroism of C-glycosylflavones

Examination of a variety of arabinose containing C-glycosylflavones has shown that the sign and intensity of the CD band at 250–275 nm (charge-transfer band) reflect not only the point of attachment of the sugar to the flavone but also depend upon the absolute and anomeric configuration, ring-size and ring-conformation in addition to the preferred rotameric conformation of the sugar about the C-aryl, C-l″ bond. A change in stereochemistry of arabinose from the α to β anomer resulted in sign inversion of the 250–275 nm CD band for 6-C-l-arabinosylflavones. Furthermore, a 6-C-arabinosylflavone containing α-l-arabinose exhibited an oppositely signed charge-transfer CD band in comparison to one which contained α-d-arabinose. 6,8-Di-C-glycosylflavones containing arabinose and glucose exhibited CD bands resulting from contributions due to both sugars, if the arabinose was not present as the β-pyranose form (¹C₄, conformation).