The effect of pH and nitrite on the haem pocket of GLB-33, a globin-coupled neuronal transmembrane receptor of Caenorhabditis elegans.

Out of the 34 globins in Caenorhabditis elegans, GLB-33 is a putative globin-coupled transmembrane receptor with a yet unknown function. The globin domain (GD) contains a particularly hydrophobic haem pocket, that rapidly oxidizes to a low-spin hydroxide-ligated haem state at physiological pH. Moreover, the GD has one of the fastest nitrite reductase activity ever reported for globins. Here, we use a combination of electronic circular dichroism, resonance Raman and electron paramagnetic resonance (EPR) spectroscopy with mass spectrometry to study the pH dependence of the ferric form of the recombinantly over-expressed GD in the presence and absence of nitrite. The competitive binding of nitrite and hydroxide is examined as well as nitrite-induced haem modifications at acidic pH. Comparison of the spectroscopic results with data from other haem proteins allows to deduce the important effect of Arg at position E10 in stabilization of exogenous ligands. Furthermore, continuous-wave and pulsed EPR indicate that ligation of nitrite occurs in a nitrito mode at pH 5.0 and above. At pH 4.0, an additional formation of a nitro-bound haem form is observed along with fast formation of a nitri-globin.

Interaction of nitrite with ferric protoglobin from Methanosarcina acetivorans - an interesting model for spectroscopic studies of the haem-ligand interaction.

Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin belonging to the same lineage of the globin superfamily as globin-coupled sensors. A putative role in the scavenging of reactive nitrogen and oxygen species has been suggested as a possible adaptation mechanism of the host organism to different gaseous environments in the course of evolution. A combination of optical absorption, electronic circular dichroism (ECD), resonance Raman (rRaman), and electron paramagnetic resonance (EPR) reveal the unusual in vitro reaction of ferric MaPgb with nitrite. In contrast to other globins, a large excess of nitrite did not induce the formation of a nitriglobin form in MaPgb. Surprisingly, the addition of nitrite in mildly acidic pH led to the formation of a stable nitric-oxide ligated ferric form of the protein (MaPgb-NO). Furthermore, the 300-700 nm ECD spectrum of ferric MaPgb is for the first time reported and discussed, showing strong differences in the Soret and Q ellipticity compared to ferric myoglobin, in line with the unusually strongly ruffled haem group of MaPgb and the related quantum-mechanical admixture of the S = 5/2 and S = 3/2 state of its ferric form. The Soret and Q ellipticity change strongly upon formation of MaPgb-NO, revealing a significant effect of the nitric-oxide ligation on the haem group and pocket. The related changes in the asymmetric pyrrole half-ring stretching vibration modes observed in the rRaman spectra give experimental support to earlier theoretical models, in which an important role of the in-plane breathing modes of the haem was predicted for the stabilization of the binding of diatomic gases to MaPgb.

GLB-3: A resilient, cysteine-rich, membrane-tethered globin expressed in the reproductive and nervous system of Caenorhabditis elegans.

The popular genetic model organism Caenorhabditis elegans (C. elegans) encodes 34 globins, whereby the few that are well-characterized show divergent properties besides the typical oxygen carrier function. Here, we present a biophysical characterization and expression analysis of C. elegans globin-3 (GLB-3). GLB-3 is predicted to exist in two isoforms and is expressed in the reproductive and nervous system. Knockout of this globin causes a 99% reduction in fertility and reduced motility. Spectroscopic analysis reveals that GLB-3 exists as a bis-histidyl-ligated low-spin form in both the ferrous and ferric heme form. A function in binding of diatomic gases is excluded on the basis of the slow CO-binding kinetics. Unlike other globins, GLB-3 is also not capable of reacting with H2O2, H2S, and nitrite. Intriguingly, not only does GLB-3 contain a high number of cysteine residues, it is also highly stable under harsh conditions (pH = 2 and high concentrations of H2O2). The resilience diminishes when the N- and C-terminal extensions are removed. Redox potentiometric measurements reveal a slightly positive redox potential (+8 ± 19 mV vs. SHE), suggesting that the heme iron may be able to oxidize cysteines. Electron paramagnetic resonance shows that formation of an intramolecular disulphide bridge, involving Cys70, affects the heme-pocket region. The results suggest an involvement of the globin in (cysteine) redox chemistry.

Vibrational Optical Activity Study of Four Antibiotic (Lipo)glycopeptides: Vancomycin, Oritavancin, Dalbavancin, and Teicoplanin.

The antibiotic glycopeptide class, of which vancomycin is the original compound, has received due attention over the past few decades in search of antibiotics to overcome resistances developed by bacteria. Crucial for the understanding and further development of glycopeptides that possess desired antibacterial effects is the determination of their conformational behavior, as this sheds light on the mechanism of action of the compound. Among others, vibrational optical activity (VOA) techniques (vibrational circular dichroism and Raman optical activity) can be deployed for this, but the question remains to what extent these spectroscopic techniques can provide information concerning the molecular class under investigation. This contribution takes the last hurdle in the search for the capabilities of the VOA techniques in the conformational analysis of the antibiotic glycopeptide class by extending research that was previously conducted for vancomycin toward its three derivatives: oritavancin, dalbavancin, and teicoplanin. The principal information that can be drawn from VOA spectra is the conformation of the rigid cyclic parts of the glycopeptides and the aromatic rings that are part hereof. The addition or removal of carbohydrates does not induce noticeable VOA spectral responses, preventing the determination of the conformation they adopt.

Insights in the vibrational optical activity spectra of the antibiotic vancomycin in DMSO.

Vibrational circular dichroism (VCD) and Raman optical activity (ROA) are two spectroscopic techniques that are sensitive towards the conformational behaviour of molecules, and are often complementary herein. In this work we pursue the determination of the conformational ensemble of the antibiotic glycopeptide vancomycin in DMSO through comparison of experimental and computational spectra, both for VCD and ROA. ROA is found to be highly suitable for the task, identifying an ensemble that strongly resembles the NMR conformation. In the case of VCD, however, a too high sensitivity of the intensities with respect to minor conformational changes hampers a reliable conformational analysis. Whence attempting to improve the match between the VCD experiment and calculations by any means - e.g., by inducing minor conformational changes or including solvent effects in the calculations - we show that there is the risk of going down the rabbit hole. In conclusion, this work contributes to the broader understanding of where, when and how VCD and ROA can be deployed as techniques for conformational analysis.

Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution.

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Tackling Stereochemistry in Drug Molecules with Vibrational Optical Activity.

Chirality plays a crucial role in drug discovery and development. As a result, a significant number of commercially available drugs are structurally dissymmetric and enantiomerically pure. The determination of the exact 3D structure of drug candidates is, consequently, of paramount importance for the pharmaceutical industry in different stages of the discovery pipeline. Traditionally the assignment of the absolute configuration of druggable molecules has been carried out by means of X-ray crystallography. Nevertheless, not all molecules are suitable for single-crystal growing. Additionally, valuable information about the conformational dynamics of drug candidates is lost in the solid state. As an alternative, vibrational optical activity (VOA) methods have emerged as powerful tools to assess the stereochemistry of drug molecules directly in solution. These methods include vibrational circular dichroism (VCD) and Raman optical activity (ROA). Despite their potential, VCD and ROA are still unheard of to many organic and medicinal chemists. Therefore, the present review aims at highlighting the recent use of VOA methods for the assignment of the absolute configuration of chiral small-molecule drugs, as well as for the structural analysis of biologics of pharmaceutical interest. A brief introduction on VCD and ROA theory and the best experimental practices for using these methods will be provided along with selected representative examples over the last five years. As VCD and ROA are commonly used in combination with quantum calculations, some guidelines will also be presented for the reliable simulation of chiroptical spectra. Special attention will be paid to the complementarity of VCD and ROA to unambiguously assess the stereochemical properties of pharmaceuticals.

Paving the way to conformationally unravel complex glycopeptide antibiotics by means of Raman optical activity.

It is crucial for fundamental physical chemistry techniques to find their application in tackling real-world challenges. Hitherto, Raman optical activity (ROA) spectroscopy is one of the examples where a promising future within the pharmaceutical sector is foreseen, but has not yet been established. Namely, the technique is believed to be able to contribute in investigating the conformational behaviour of drug candidates. We, herein, strive towards the alignment of the ROA analysis outcome and the pharmaceutical expectations by proposing a fresh strategy that ensures a more complete, reliable, and transferable ROA study. The strategy consists of the treatment of the conformational space by means of a principal component analysis (PCA) and a clustering algorithm, succeeded by a thorough ROA spectral analysis and a novel way of estimating the contributions of the different chemical fragments to the total ROA spectral intensities. Here, vancomycin, an antibiotic glycopeptide, has been treated; it is the first antibiotic glycopeptide studied by means of ROA and is a challenging compound in ROA terms. By applying our approach we discover that ROA is capable of independently identifying the correct conformation of vancomycin in aqueous solution. In addition, we have a clear idea of what ROA can and cannot tell us regarding glycopeptides. Finally, the glycopeptide class turns out to be a spectroscopically curious case, as its spectral responses are unlike the typical ROA spectral responses of peptides and carbohydrates. This preludes future ROA studies of this intriguing molecular class.

Employing complementary spectroscopies to study the conformations of an epimeric pair of side-chain stapled peptides in aqueous solution.

Understanding the conformational preferences of free ligands in solution is often necessary to rationalize structure-activity relationships in drug discovery. Herein, we examine the conformational behavior of an epimeric pair of side-chain stapled peptides that inhibit the FAD dependent amine oxidase lysine specific demethylase 1 (LSD1). The peptides differ only at a single stereocenter, but display a major difference in binding affinity. Their Raman optical activity (ROA) spectra are most likely dominated by the C-terminus, obscuring the analysis of the epimeric macrocycle. By employing NMR spectroscopy, we show a difference in conformational behavior between the two compounds and that the LSD1 bound conformation of the most potent compound is present to a measurable extent in aqueous solution. In addition, we illustrate that Molecular Dynamics (MD) simulations produce ensembles that include the most important solution conformations, but that it remains problematic to identify relevant conformations with no a priori knowledge from the large conformational pool. Furthermore, this work highlights the importance of understanding the scope and limitations of the available techniques for conducting conformational analyses. It also emphasizes the importance of conformational selection of a flexible ligand in molecular recognition.

Metal ions shape α-synuclein.

α-Synuclein is an intrinsically disordered protein that can self-aggregate and plays a major role in Parkinson's disease (PD). Elevated levels of certain metal ions are found in protein aggregates in neurons of people suffering from PD, and environmental exposure has also been linked with neurodegeneration. Importantly, cellular interactions with metal ions, particularly Ca2+, have recently been reported as key for α-synuclein's physiological function at the pre-synapse. Here we study effects of metal ion interaction with α-synuclein at the molecular level, observing changes in the conformational behaviour of monomers, with a possible link to aggregation pathways and toxicity. Using native nano-electrospray ionisation ion mobility-mass spectrometry (nESI-IM-MS), we characterize the heterogeneous interactions of alkali, alkaline earth, transition and other metal ions and their global structural effects on α-synuclein. Different binding stoichiometries found upon titration with metal ions correlate with their specific binding affinity and capacity. Subtle conformational effects seen for singly charged metals differ profoundly from binding of multiply charged ions, often leading to overall compaction of the protein depending on the preferred binding sites. This study illustrates specific effects of metal coordination, and the associated electrostatic charge patterns, on the complex structural space of the intrinsically disordered protein α-synuclein.

A combined Raman optical activity and vibrational circular dichroism study on artemisinin-type products.

Artemisinin and two of its derivatives, dihydroartemisinin and artesunate, which are front line drugs against malaria, were investigated using Raman optical activity (ROA) and vibrational circular dichroism (VCD) experiments, both supported by density functional theory (DFT) level calculations. The experimental techniques combined with DFT calculations could show that dihydroartemisinin was present as an epimeric mixture in solution. In addition, an approximation of the epimeric ratio could be extracted which was in agreement with the ratio obtained by 1H-NMR spectroscopy. The current study also demonstrates that both ROA and VCD are able to assign the correct absolute configuration (AC) of artemisinin and artesunate out of all their possible diastereomers without any explicit knowledge on their correct stereochemistry and accentuates the synergetic effect between ROA and VCD in AC determination.

Comparative Study of the Vibrational Optical Activity Techniques in Structure Elucidation: The Case of Galantamine.

The absolute configuration of the alkaloid galantamine was studied using a range of solution-state techniques; nuclear magnetic resonance (NMR), vibrational circular dichroism (VCD), and Raman optical activity (ROA). While the combined use of NMR and VCD does provide a fast, high-resolution methodology for determining the absolute configuration of galantamine, both techniques were needed in concert to achieve this goal. ROA, on the other hand, proved to be sensitive enough to assign the full absolute configuration without relying on other techniques. In both cases, statistical validation was applied to aid the determination of absolute configuration. In the case of galantamine, ROA combined with statistical validation is shown to be a powerful stand-alone tool for absolute configuration determination.

Effects of sulfation and the environment on the structure of chondroitin sulfate studied via Raman optical activity.

Glycosaminoglycans are linear carbohydrate polymers with essential roles in many biological processes. Chondroitin sulfate (CS) is one of them, omnipresent in living organisms as an important structural component of cartilage. It provides much of its resistance to compression. Despite its biological importance, little is still known about the relation of the CS structure to chemical composition and interaction with the environment. We therefore measured Raman and Raman optical activity (ROA) spectra of five CS samples of different biological origin and variously sulfated CS building blocks (GlcA, GalNAc, and basic disaccharide units) in a wide frequency range between 200 cm-1 and 1800 cm-1 and analyzed them with respect to specific structure marker bands. We show that ROA spectroscopy is sensitive to the conformational stability and rigidity of pyranose rings of saccharides, the orientation of sugar hydroxyl groups and the secondary structure of the CS's backbone. The CS secondary structure has been found to be quite stable, with a minor variation as a reaction to physicochemical parameters (concentration, pH, temperature, and the presence of cations). Larger changes were observed under chemical changes (sulfation) of the CS chain. ROA spectroscopy thus exhibited useful potential to study the structure of similar biopolymers.

The effect of protein backbone hydration on the amide vibrations in Raman and Raman optical activity spectra.

Raman and specifically Raman optical activity (ROA) spectroscopy are very sensitive to the solution structure and conformation of biomolecules. Because of this strong conformational sensitivity, density functional theory (DFT) calculations are often used to get a better understanding of the experimentally observed spectral patterns. While e.g. for carbohydrate structure the water molecules that surround the solute have been demonstrated to be of vital importance to get accurate modelled ROA spectra, the effect of explicit water molecules on the calculated ROA patterns of peptides and proteins is less well studied. Here, the effect of protein backbone hydration was studied using DFT calculations of HCO-(l-Ala)5-NH2 in specific secondary structure conformations with different treatments of the solvation. The effect of the explicit water molecules on the calculated spectra mainly arises from the formation of hydrogen bonds with the amide C[double bond, length as m-dash]O and N-H groups. Hydrogen bonding of water with the C[double bond, length as m-dash]O group determines the shape and position of the amide I band. The C[double bond, length as m-dash]O bond length increases upon formation of C[double bond, length as m-dash]OH2O hydrogen bonds. The effect of the explicit water molecules on the amide III vibrations arises from hydrogen bonding of the solvent with both the C[double bond, length as m-dash]O and N-H group, but their contributions to this spectral region differ: geometrically, the formation of a C[double bond, length as m-dash]OH2O bond decreases the C-N bond length, while upon forming a N-HH2O hydrogen bond, the N-H bond length increases.

Solution Structure of Mannobioses Unravelled by Means of Raman Optical Activity.

Structural analysis of carbohydrates is a complicated endeavour, due to the complexity and diversity of the samples at hand. Herein, we apply a combined computational and experimental approach, employing molecular dynamics (MD) and density functional theory (DFT) calculations together with NMR and Raman optical activity (ROA) measurements, in the structural study of three mannobiose disaccharides, consisting of two mannoses with varying glycosidic linkages. The disaccharide structures make up the scaffold of high mannose glycans and are therefore important targets for structural analysis. Based on the MD population analysis and NMR, the major conformers of each mannobiose were identified and used as input for DFT analysis. By systematically varying the solvent models used to describe water interacting with the molecules and applying overlap integral analysis to the resulting calculational ROA spectra, we found that a full quantum mechanical/molecular mechanical approach is required for an optimal calculation of the ROA parameters. Subsequent normal mode analysis of the predicted vibrational modes was attempted in order to identify possible marker bands for glycosidic linkages. However, the normal mode vibrations of the mannobioses are completely delocalised, presumably due to conformational flexibility in these compounds, rendering the identification of isolated marker bands unfeasible.

Tracking Conformational Changes in Phosvitin throughout a Crowding-Agent-Based Titration.

The sensitivity of Raman optical activity (ROA) towards small conformational changes is explored by tracking the structural changes in an intrinsically disordered protein-phosvitin-induced by different concentrations of crowding agent. It is shown that ROA is capable of tracking small conformational changes involving ß-sheet and α-helical secondary structural properties of the protein. Furthermore, it is indicated that the influences of the crowding agents employed, Ficoll 70 and dextran 70, on the structural properties of phosvitin differ significantly, with the structural changes induced by the presence of Ficoll 70 being more pronounced and already being visible at a lower concentration. The data also suggest that some spectral changes do not arise from a change in the secondary structure of the protein, but are related to differences in interaction between the phosphorylated residues of the protein and the sugar-based crowding agent.

The Influence of the Amino Acid Side Chains on the Raman Optical Activity Spectra of Proteins.

The Raman optical activity (ROA) spectra of proteins show distinct patterns arising from the secondary structure. It is generally believed that the spectral contributions of the side-chains largely cancel out because of their flexibility and the occurrence of many side-chains with different conformations. Yet, the influence of the side-chains on the ROA patterns assigned to different secondary structures is unknown. Here, the first systematic study of the influence of all amino acid side-chains on the ROA patterns is presented based on density functional theory (DFT) calculations of an extensive collection of peptide models that include many different side-chain and secondary structure conformations. It was shown that the contributions of the side-chains to a large extent average out with conformational flexibility. However, specific side-chain conformations can have significant contributions to the ROA patterns. It was also shown that α-helical structure is very sensitive to both the exact backbone conformation and the side-chain conformation. Side-chains with χ1 ≈-60° generate ROA patterns alike those in experiment. Aromatic side-chains strongly influence the amide III ROA patterns. Because of the huge structural sensitivity of ROA, the spectral patterns of proteins arise from extensive conformational averaging of both the backbone and the side-chains. The averaging results in the fine spectral details and relative intensity differences observed in experimental spectra.

ß2-Type Amyloidlike Fibrils of Poly-l-glutamic Acid Convert into Long, Highly Ordered Helices upon Dissolution in Dimethyl Sulfoxide.

Replacing water with dimethyl sulfoxide (DMSO) completely reshapes the free-energy landscapes of solvated proteins. In DMSO, a powerful hydrogen-bond (HB) acceptor, formation of HBs between backbone NH groups and solvent is favored over HBs involving protein's carbonyl groups. This entails a profound structural disruption of globular proteins and proteinaceous aggregates (e.g., amyloid fibrils) upon transfer to DMSO. Here, we investigate an unusual DMSO-induced conformational transition of ß2-amyloid fibrils from poly-l-glutamic acid (PLGA). The infrared spectra of ß2-PLGA dissolved in DMSO lack the typical features associated with disordered conformation that are observed when amyloid fibrils from other proteins are dispersed in DMSO. Instead, the frequency and unusual narrowness of the amide I band imply the presence of highly ordered helical structures, which is supported by complementary methods, including vibrational circular dichroism and Raman optical activity. We argue that the conformation most consistent with the spectroscopic data is that of a PLGA chain essentially lacking nonhelical segments such as bends that would provide DMSO acceptors with direct access to the backbone. A structural study of DMSO-dissolved ß2-PLGA by synchrotron small-angle X-ray scattering reveals the presence of long uninterrupted helices lending direct support to this hypothesis. Our study highlights the dramatic effects that solvation may have on conformational transitions of large polypeptide assemblies.

Vibrational Circular Dichroism Sheds New Light on the Competitive Effects of Crowding and ß-Synuclein on the Fibrillation Process of α-Synuclein.

The effects of crowding, using the crowding agent Ficoll 70, and the presence of ß-synuclein on the fibrillation process of α-synuclein were studied by spectroscopic techniques, transmission electron microscopy, and thioflavin T assays. This combined approach, in which all techniques were applied to the same original sample, generated an unprecedented understanding of the effects of these modifying agents on the morphological properties of the fibrils. Separately, crowding gives rise to shorter mutually aligned fibrils, while ß-synuclein leads to branched, short fibrils. The combination of both effects leads to short, branched, mutually aligned fibrils. Moreover, it is shown that the nondestructive technique of vibrational circular dichroism is extremely sensitive to the length and the higher-order morphology of the fibrils.

Is Raman Optical Activity Spectroscopy Sensitive to ß-Turns in Proteins? Secondary Structure and Side-Chain Dependence.

ß-turns are essential for the structure and function of proteins. The spectroscopic technique Raman optical activity (ROA) has been suggested to be sensitive to such structural elements of proteins in solution. Three spectral features have been reported to mark ß-turns in protein ROA spectra: being a negative band at 1220 cm-1 , positive intensity around 1290 cm-1 and negative intensity around 1340-1380 cm-1 . In this work, density functional theory calculations demonstrated that these assignments are inaccurate as these spectral regions are not robust and sense the exact secondary structure surrounding the ß-turn as well. Furthermore, it was demonstrated that the amino acid side-chains affect the exact ROA patterns which can direct future research to perform a systematic analysis of the contributions of the side-chains.