Tools and methods for circular dichroism spectroscopy of proteins: a tutorial review.

Circular dichroism (CD) spectroscopy is a widely-used method in biochemistry, structural biology and pharmaceutical chemistry. More than 24 000 papers published in the past decade have included CD characterisations of proteins; many of those studies have also included other complementary chemical, biophysical, and computational chemistry methods. This tutorial review describes the background to the technique of CD spectroscopy and good practice methods for high quality data collection. It specifically focuses on both established and new methods and tools available for experimental design and interpretation, data processing, visualisation, analysis, validation, archiving, and accession, including tools developed to enhance the complementarity of this method with other structural and chemical biology studies.

Dynamically adjustable-induced THz circular dichroism and biosensing application of symmetric silicon-graphene-metal composite nanostructures.

Induced circular dichroism (ICD) has been used to detect biomolecular conformations through the coupling between chiral molecules and achiral metal nanostructures with the localized surface plasmon (LSP). However, this ICD is always weak and cannot be dynamically adjusted. Here, we put dielectric and graphene nanostructures on a metal-substrate for restricting more light energies and obtaining dynamic adjustable performance. A composite nanostructure array composed of achiral silicon-nanorods on a metal-substrate and graphene-ribbons (ASMG) is theoretically investigated. Two strong ICD signals appear in the THz region. Near-field magnetic distributions of ASMG reveal that the two strong ICD signals are mainly due to the surface plasmon resonances (SPPs) on the metal-substrate and LSP in the graphene nanostructures, respectively. The ICD signals strongly depend on the geometric parameters of ASMG and are dynamically adjusted by just changing the Fermi levels of graphene-ribbons. In addition, left-handed ASMG and right-handed ASMG can be used to identify the chiral molecular solutions with different chiralities. The maximum enhancement factor of the chiral molecular solutions could reach up to 3500 times in the THz region. These results can help to design dynamically adjustable THz chiral sensors and promote their application in biological monitoring and asymmetric catalysis.

Analytical and Structural Studies for the Investigation of Oxidative Stress in Guanine Oligonucleotides.

DNA damage plays a decisive role in epigenetic effects. The detection and analysis of DNA damages, like the most common change of guanine (G) to 8-oxo-7,8-dihydroguanine (OG), is a key factor in cancer research. It is especially true for G quadruplex structure (GQ), which is one of the best-known examples of a non-canonical DNA arrangement. In the present work, we provided an overview on analytical methods in connection with the detection of OG in oligonucleotides with GQ-forming capacity. Focusing on the last five years, novel electrochemical tools, like dedicated electrodes, were overviewed, as well as different optical methods (fluorometric assays, resonance light scattering or UV radiation) along with hyphenated detection and structural analysis methods (CD, NMR, melting temperature analysis and nanopore detection) were also applied for OG detection. Additionally, GQ-related computational simulations were also summarized. All these results emphasize that OG detection and the analysis of the effect of its presence in higher ordered structures like GQ is still a state-of-the-art research line with continuously increasing interest.

Application of Synchrotron Radiation Circular Dichroism for RNA Structural Analysis.

Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170 nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.

Automated High-Throughput Capillary Circular Dichroism and Intrinsic Fluorescence Spectroscopy for Rapid Determination of Protein Structure.

Assessing the physical stability of proteins is one of the most important challenges in the development, manufacture, and formulation of biotherapeutics. Here, we describe a method for combining and automating circular dichroism and intrinsic protein fluorescence spectroscopy. By robotically injecting samples from a 96-well plate into an optically compliant capillary flow cell, complementary information about the secondary and tertiary structural state of a protein can be collected in an unattended manner from considerably reduced volumes of sample compared to conventional techniques. We demonstrate the accuracy and reproducibility of this method. Furthermore, we show how structural screening can be used to monitor unfolding of proteins in two case studies using (i) a chaotropic denaturant (urea) and (ii) low-pH buffers used for monoclonal antibody (mAb) purification during Protein A chromatography.

Coupled Binding and Helix Formation Monitored by Synchrotron-Radiation Circular Dichroism.

Intrinsically disordered proteins organize interaction networks in the cell in many regulation and signaling processes. These proteins often gain structure upon binding to their target proteins in multistep reactions involving the formation of both secondary and tertiary structure. To understand the interactions of disordered proteins, we need to understand the mechanisms of these coupled folding and binding reactions. We studied helix formation in the binding of the molten globule-like nuclear coactivator binding domain and the disordered interaction domain from activator of thyroid hormone and retinoid receptors. We demonstrate that helix formation in a rapid binding reaction can be followed by stopped-flow synchrotron-radiation circular dichroism (CD) spectroscopy and describe the design of such a beamline. Fluorescence-monitored binding experiments of activator of thyroid hormone and retinoid receptors and nuclear coactivator binding domain display several kinetic phases, including one concentration-independent phase, which is consistent with an intermediate stabilized at high ionic strength. Time-resolved CD experiments show that almost all helicity is formed upon initial association of the proteins or separated from the encounter complex by only a small energy barrier. Through simulation of mechanistic models, we show that the intermediate observed at high ionic strength likely involves a structural rearrangement with minor overall changes in helicity. Our experiments provide a benchmark for simulations of coupled binding reactions and demonstrate the feasibility of using synchrotron-radiation CD for mechanistic studies of protein-protein interactions.

Refining Calibration Procedures of Circular Dichroism Spectrometer to Improve Usability.

Circular dichroism (CD) is a technique used for conformational studies of peptides and proteins. We studied the specific calibration procedures of CD spectrometers based on procedures specified in the European Pharmacopoeia. We aimed to develop procedures to improve the usability of CD, in addition to reducing adverse effects on users' health. The use of ethanol instead of 1,4-dioxane as the solvent for isoandrosterone was examined. Both solvents yielded the same maximum value of +3.3 for molar CD. We also studied a two-point calibration method using (1S)-(+)-ammonium 10-camphorsulfonate instead of (1S)-(+)-10-camphorsulfonic acid, which is a hygroscopic compound. Both compounds yielded similar results and the values for (1S)-(+)-ammonium 10-camphorsulfonate of 2.39 ± 0.04 and -4.92 ± 0.06 at 290.5 and 192.5 nm, respectively, were within the criteria defined in the European Pharmacopoeia. The inter-laboratory repeatability was also acceptable. These studies provide specific procedures for calibrating CD spectrometers for drug development.

Biomimetic Chiral Photonic Crystals.

Although it is well known that the amazing iridescent colors of the cuticle of beetles reflect the intricate nanoscale organization of bio-fibers, artificial inorganic materials with comparable optical responses have not yet been synthesized from abiotic nanoscale building blocks. Such materials could find broad applications, including in circular polarizers, to generate circularly polarized luminescence, or in lasers. Herein, we describe a general method for the fabrication of biomimetic chiral photonic crystals by Langmuir-Schaefer assembly of colloidal inorganic nanowires. We not only reproduced the intricate helical structure and circularly polarized color reflection observed in beetles, but also achieved the highest chiroptical activity with a dissymmetry factor of -1.6 ever reported for chiral inorganic nanostructures. More importantly, the programmable structural control based on the precise interlayer arrangement endows us with unprecedented freedom to manipulate the optical activity of as-fabricated chiral photonic crystals.

Highly selective circular dichroism sensor based on d-penicillamine/cysteamine­cadmium sulfide quantum dots for copper (II) ion detection.

A highly selective circular dichroism sensor based on cysteamine-capped cadmium sulfide quantum dots (Cys-CdS QDs) was successfully developed for the determination of Cu2+. Basically, the Cys-CdS QDs are not active in circular dichroism spectroscopy. However, the circular dichroism probe (DPA@Cys-CdS QDs) can be simply generated in situ by mixing achiral Cys-CdS QDs with D-penicillamine. The detection principle was based on measuring the circular dichroism signal change upon the addition of Cu2+. The strong CD signal of the DPA@Cys-CdS QDs dramatically decreased in the presence of Cu2+, while other cations did not result in any spectral changes. In addition, the degree of the CD signal decrease was linearly proportional to the concentration of Cu2+ in the range of 0.50-2.25 µM (r2 = 0.9919) with a detection limit of 0.34 µM. Furthermore, the proposed sensor was applied to detect Cu2+ in drinking water samples with %recovery values of 102-114% and %RSD less than 6%.

STRUCTURAL ANALYSIS OF DNA REPAIR PROTEIN XRCC4 APPLYING CIRCULAR DICHROISM IN AN AQUEOUS SOLUTION.

We applied circular dichroism (CD) spectral analysis to obtain the secondary structure of the DNA repair protein XRCC4, with a focus on its C-terminus. Using synchrotron radiation as a light source across a wide range of wavelengths, including vacuum ultraviolet (UV) light from 180 to 200 nm and UV light from 200 to 240 nm, we determined the secondary structure composition of the full-length protein, including its C-terminus, which had not yet been -the focus of crystallography studies, though it contains several phosphorylation sites. The secondary structures inferred using the obtained CD spectra indicate that the C-terminus is composed of a substantial fraction of turns with a few unordered and alpha-helix structures and almost no beta-strands. The C-terminus is likely to form a characteristic secondary structure with turns as a main component, and its DNA repair function is likely regulated by the structural change induced by phosphorylation of the terminus.

Resolving protein mixtures using microfluidic diffusional sizing combined with synchrotron radiation circular dichroism.

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.

Discovery of the first self-assembling peptide, study of peptide dynamic behaviors, and G protein-coupled receptors using an Aviv circular dichroism spectropolarimeter.

Circular dichroism (CD) spectroscopy is a useful technique to study the structure and dynamics of peptides, proteins and nucleic acids. CD is particularly useful because sample volumes may be as low as 50 µL, it provides high precision and sensitivity, and it achieves a good signal to noise ratio. CD characterizes molecular conformational changes in real time by finely controlling temperature, pH, and titrating urea and guanidine·HCl which is necessary for studying protein folding. Although CD does not provide detailed structure at the atomic level, it provides a global structural framework. Researchers use CD to observe molecular phenomena, namely how macromolecules unfold/refold and their overall self-assembly/disassembly. Using CD to monitor a peptide structure, I serendipitously discovered the self-assembling peptide EAK16 from yeast protein Zuotin. This unusual peptide formed a new type of nanofiber scaffold hydrogel material. The discovery in 1990 opened a new field in the design and study of numerous self-assembling peptides, thereby launching the area of peptide nanobiotechnology. In this review, I reflect on my personal discoveries of several self-assembling peptides, investigations into the dynamic behaviors of peptides, as well as the impact of the work on society. I also describe studies of natural membrane proteins and engineered membrane proteins using CD. Furthermore, I enjoyed numerous and close interactions with Jack Aviv since 1997. He generously supported 10 high impact workshops (Crete and Mikonos) and meetings in various countries around the world that left fond memories of many young researches who later became leading scientists in their respective fields.

Instrumentation for Vibrational Circular Dichroism Spectroscopy: Method Comparison and Newer Developments.

Vibrational circular dichroism (VCD) is a widely used standard method for determination of absolute stereochemistry, and somewhat less so for biomolecule characterization and following dynamic processes. Over the last few decades, different VCD instrument designs have developed for various purposes, and reliable commercial instrumentation is now available. This review will briefly survey historical and currently used instrument designs and describe some aspects of more recently reported developments. An important factor in applying VCD to conformational studies is theoretical modeling of spectra for various structures, techniques for which are briefly surveyed.

Near shot-noise limited time-resolved circular dichroism pump-probe spectrometer.

We describe an optical near shot-noise limited time-resolved circular dichroism (TRCD) pump-probe spectrometer capable of reliably measuring circular dichroism signals in the order of µdeg with nanosecond time resolution. Such sensitivity is achieved through a modification of existing TRCD designs and introduction of a new data processing protocol that eliminates approximations that have caused substantial nonlinearities in past measurements and allows the measurement of absorption and circular dichroism transients simultaneously with a single pump pulse. The exceptional signal-to-noise ratio of the described setup makes the TRCD technique applicable to a large range of non-biological and biological systems. The spectrometer was used to record, for the first time, weak TRCD kinetics associated with the triplet state energy transfer in the photosynthetic Fenna-Matthews-Olson antenna pigment-protein complex.

Measuring fluorescence polarization with a dichrometer.

A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer.

Advantages of synchrotron radiation circular dichroism spectroscopy to study intrinsically disordered proteins.

The unordered secondary structural content of an intrinsically disordered protein (IDP) is susceptible to conformational changes induced by many different external factors, such as the presence of organic solvents, removal of water, changes in temperature, binding to partner molecules, and interaction with lipids and/or other ligands. In order to characterize the high-flexibility nature of an IDP, circular dichroism (CD) spectroscopy is a particularly useful method due to its capability of monitoring both subtle and remarkable changes in different environments, relative ease in obtaining measurements, the small amount of sample required, and the capability for sample recovery (sample not damaged) and others. Using synchrotron radiation as the light source for CD spectroscopy represents the state-of-the-art version of this technique with feasibility of accessing the lower wavelength UV region, and therefore presenting a series of advantages over conventional circular dichroism (cCD) to monitor a protein conformational behavior, check protein stability, detect ligand binding, and many others. In this paper, we have performed a comparative study using cCD and SRCD methods for investigating the secondary structure and the conformational behavior of natively unfolded proteins: MEG-14 and soybean trypsin inhibitor. We show that the SRCD technique greatly improves the analysis and accuracy of the studies on the conformations of IDPs.

Circular Dichroism and Fluorescence Spectroscopy to Study Protein Structure and Protein-Protein Interactions in Ethylene Signaling.

Circular dichroism (CD) spectroscopy is an invaluable technique to analyze secondary structure and functional folding of recombinant purified proteins. CD spectroscopy can also be applied to detect changes in protein secondary structure related to the pH or redox conditions found in different cellular compartments or to the interaction with other molecules. Another biophysical technique to monitor conformational changes and interaction with small molecule ligands or biological macromolecules is protein fluorescence spectroscopy making use of the aromatic amino acid tryptophan as a sensitive intrinsic fluorescent probe. Here, we describe the application of CD and tryptophan fluorescence spectroscopy to study soluble and membrane proteins of the ethylene signaling pathway.

High-throughput SRCD using multi-well plates and its applications.

The sample compartment for high-throughput synchrotron radiation circular dichroism (HT-SRCD) has been developed to satisfy an increased demand of protein characterisation in terms of folding and binding interaction properties not only in the traditional field of structural biology but also in the growing research area of material science with the potential to save time by 80%. As the understanding of protein behaviour in different solvent environments has increased dramatically the development of novel functions such as recombinant proteins modified to have different functions from harvesting solar energy to metabolonics for cleaning heavy and metal and organic molecule pollutions, there is a need to characterise speedily these system.

Characterisation of Conformational and Ligand Binding Properties of Membrane Proteins Using Synchrotron Radiation Circular Dichroism (SRCD).

Membrane proteins are notoriously difficult to crystallise for use in X-ray crystallographic structural determination, or too complex for NMR structural studies. Circular dichroism (CD) is a fast and relatively easy spectroscopic technique to study protein conformational behaviour in solution. The advantage of synchrotron radiation circular dichroism (SRCD) measured with synchrotron beamlines compared to the CD from benchtop instruments is the extended spectral far-UV region that increases the accuracy of secondary structure estimations, in particular under high ionic strength conditions. Membrane proteins are often available in small quantities, and for this SRCD measured at the Diamond B23 beamline has successfully facilitated molecular recognition studies. This was done by probing the local tertiary structure of aromatic amino acid residues upon addition of chiral or non-chiral ligands using long pathlength cells (1-5 cm) of small volume capacity (70 µl-350 µl). In this chapter we describe the use of SRCD to qualitatively and quantitatively screen ligand binding interactions (exemplified by Sbma, Ace1 and FsrC proteins); to distinguish between functionally similar drugs that exhibit different mechanisms of action towards membrane proteins (exemplified by FsrC); and to identify suitable detergent conditions to observe membrane protein-ligand interactions using stabilised proteins (exemplified by inositol transporters) as well as the stability of membrane proteins (exemplified by GalP, Ace1). The importance of the in solution characterisation of the conformational behaviour and ligand binding properties of proteins in both far- andnear-UV regions and the use of high-throughput CD (HT-CD) using 96- and 384-well multiplates to study the folding effects in various protein crystallisation buffers are also discussed.

Estudos biofísicos e de atividade de peptídeos correspondentes ao N-terminal das toxinas esticolisinais I e II - contribuição para a elucidação do mecanismo de ação / Biophysical and activity studies of peptides corresponding to the N-termini of sticholysins I and II - a contribution to the elucidation of the toxins' mechanism of action

Esticolisinas I e II, citolisinas purificadas da anêmona marinha Stichodactyla helianthus, agem lisando membranas biológicas e modelo. O mecanismo de ação proposto consiste na formação de um poro toroidal com o envolvimento do domínio N-terminal. Diferentes aspectos da interação entre peptídeos derivados do N-terminal das toxinas (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, e StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) com membranas modelo - micelas e bicamadas - foram estudados com o objetivo de contribuir para a elucidação do mecanismo de ação das toxinas em nível molecular. O emprego dos peptídeos teve como base a hipótese de que fragmentos proteicos podem ser capazes de mimetizar a estrutura e atividade das proteínas inteiras. O análogo contendo o aminoácido paramagnético TOAC (N-TOAC-StII11-30) também foi estudado. Estudos conformacionais foram realizados empregando-se as técnicas espectroscópicas de dicroísmo circular (CD), ressonância paramagnética eletrônica (EPR) e fluorescência. Foram ainda realizados estudos de predição de estrutura e modelagem molecular. Espectros de CD mostraram que os peptídeos adquirem conformação em α-hélice ao interagir com membranas modelo, de acordo com a conformação observada nessa região para as toxinas. Variando a composição lipídica das membranas modelo estudadas, foi possível investigar a contribuição de forças eletrostáticas de de interações hidrofóbicas para a ligação do peptídeo. Ensaios de supressão de fluorescência de lípidos contendo grupamentos fluorescentes em diferentes posições pelo resíduo paramagnético TOAC e espectros de ressonância paramagnética eletrônica (EPR) permitiram localizar o resíduo TOAC na interface membrana-água, corroborando o modelo proposto do poro toroidal. A análise dos espectros de CD e EPR também permitiu obter as constantes de ligação dos peptídeos com micelas e bicamadas. Os peptídeos também foram capazes de mimetizar as toxinas do ponto de vista funcional, como mostrado por testes de vazamento de carboxifluoresceína e atividade hemolítica. Peptídeos curtos, contendo partes da sequência de StII1-30, sintetizados com o objetivo de examinar uma eventual atividade antimicrobiana, demonstraram baixa atividade, bem como ausência de atividade hemolítica e de toxicidade para células humanas

Sticholysins I and II, cytolysins purified from the sea anemone Stichodactyla helianthus, act by lysing biological and model membranes. The proposed mechanism of action consists in the formation of a toroidal pore with the involvement of the N-terminal domain [1]. Different aspects of the interaction between peptides from the toxins' N-termini (StI1-31 and StI12-31 SELAGTIIDGASLTFEVLDKVLGELGKVSRK, and StII1-30 and StII11-30 ALAGTIIAGASLTFQVLDKVLEELGKVSRK) and model membranes - micelles and bilayers - have been studied to contribute to the elucidation of the toxins mechanism of action at the molecular level. The use of peptides was based on the hypothesis that potein fragments can eventually mimic the structure and activity of the whole protein. An analogue containing the paramagnetic amino acid TOAC (N-TOAC-StII11-30) was also studied. Conformational studies were performed making use of the spectroscopic techniques circular dichroism (CD), electron paramagnetic resonance (EPR), and fluorescence. Studies of structure prediction and molecular modeling were also performed. CD spectra showed that the peptides acquired α-helical conformation upon interaction with model lipid membranes, in agreement with the conformation found for these segments in the whole proteins. Making use of membranes of variable lipid composition, it was possible to assess the contribution of electrostatic and hydrophobic interactions for peptide binding. Fluorescence quenching of labeled lipids by paramagnetic TOAC and EPR spectra allowed us to locate the TOAC residue at the membrane-water interface, corroborating the proposed model of the toroidal pore. The CD and EPR studies also allowed us to obtain the binding constants for the peptide-micelle and peptide-bilayer interaction. The peptides were also capable of mimicking the toxins function, as shown by assays of carboxyfluorescein leakage and hemolytic activity. Short peptides containing parts of StII1-30's sequence were synthesized with the aim of testing their antimicrobial activity. The peptides displayed low antimicrobial activity, as well as lack of hemolytic activity and toxicity against human cells