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.

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.

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.

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.

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.

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.

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.

Circularly polarized near-field optical mapping of spin-resolved quantum Hall chiral edge states.

We have successfully developed a circularly polarized near-field scanning optical microscope (NSOM) that enables us to irradiate circularly polarized light with spatial resolution below the diffraction limit. As a demonstration, we perform real-space mapping of the quantum Hall chiral edge states near the edge of a Hall-bar structure by injecting spin polarized electrons optically at low temperature. The obtained real-space mappings show that spin-polarized electrons are injected optically to the two-dimensional electron layer. Our general method to locally inject spins using a circularly polarized NSOM should be broadly applicable to characterize a variety of nanomaterials and nanostructures.

UV-CD12: synchrotron radiation circular dichroism beamline at ANKA.

Synchrotron radiation circular dichroism (SRCD) is a rapidly growing technique for structure analysis of proteins and other chiral biomaterials. UV-CD12 is a high-flux SRCD beamline installed at the ANKA synchrotron, to which it had been transferred after the closure of the SRS Daresbury. The beamline covers an extended vacuum-UV to near-UV spectral range and has been open for users since October 2011. The current end-station allows for temperature-controlled steady-state SRCD spectroscopy, including routine automated thermal scans of microlitre volumes of water-soluble proteins down to 170 nm. It offers an excellent signal-to-noise ratio over the whole accessible spectral range. The technique of oriented circular dichroism (OCD) was recently implemented for determining the membrane alignment of α-helical peptides and proteins in macroscopically oriented lipid bilayers as mimics of cellular membranes. It offers improved spectral quality <200 nm compared with an OCD setup adapted to a bench-top instrument, and accelerated data collection by a factor of ∼3. In addition, it permits investigations of low hydrated protein films down to 130 nm using a rotatable sample cell that avoids linear dichroism artifacts.

Improved DNA equilibrium binding affinity determinations of platinum(II) complexes using synchrotron radiation circular dichroism.

The binding affinity of a series of square planar platinum(II) compounds of the type [Pt(A(L))(I(L))](2+), where A(L) is 1,2-diaminoethane and I(L) are 1,10-phenanthroline (phen), 4-methyl-1,10-phenanthroline (4Mephen), 5-methyl-1,10-phenanthroline (5Mephen), 4,7-dimethyl-1,10-phenanthroline (47Me2phen), 5,6-dimethyl-1,10-phenanthroline (56Me2phen) or 3,4,7,8-tetramethyl-1,10-phenanthroline (3478Me4phen) has been reinvestigated using Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy. The additional peaks exhibited considerably greater intensity than those observed between 200 and 400 nm affording additional binding affinity determinations. In addition, the authors have reviewed the various mathematical approaches used to estimate equilibrium binding constants and thereby demonstrate that their mathematical approach, implemented with Wolfram Mathematica, has merit over other methods.

Automated circular dichroism spectroscopy for medium-throughput analysis of protein conformation.

Circular dichroism (CD) spectroscopy is a powerful method for monitoring conformational changes of biomolecules. For peptides and proteins, it is highly sensitive to changes in secondary structure, which may be caused by alterations in amino acid composition or solution conditions (e.g., temperature, pH, salts, detergents, denaturants, and excipients), post-translational modifications, self-association, or ligand binding. The assets of CD spectroscopy are that the signal is directly linked to structure, the analyte is measured without labels and in solution, the technique requires low sample amounts, and data analysis is straightforward. However, CD spectroscopy has remained a low-throughput method because it imposes high requirements on the optical quality of sample cells and thus cannot be performed in microplate-reader format. Here, we introduce an automated CD spectrometer equipped with a low-birefringence flow-through cell that is coupled to a three-axis robotic liquid-handling system. This enables unattended CD measurements on up to 384 samples, including sample transfer from 96-well plates into the flow-through cell, data acquisition, and cell cleaning. We show that the accuracy, precision, and reproducibility afforded by the new instrument are excellent and exemplify how the advantages offered by automated CD spectroscopy can be exploited to quantify protein stability by titration with chemical denaturants.

Light shutter using dichroic-dye-doped long-pitch cholesteric liquid crystals.

We propose a light shutter device using dichroic-dye-doped liquid crystals (LCs) whose Bragg reflection wavelength is set to be infrared by controlling the pitch of cholesteric liquid crystals (ChLCs). A dye-doped long-pitch ChLC cell is switchable between the dark planar state and the transparent homeotropic state. It has the advantages of high transmittance, low operation voltage, and an easy fabrication process relative to previous LC light shutter devices. The proposed light shutter device is expected to achieve high visibility for transparent organic light-emitting diode displays and emerging smart windows, which can be used in airplanes, cars, and other similar applications.

A dichroic surface-plasmon-polariton splitter based on an asymmetric T-shape nanoslit.

An asymmetric T-shape nanoslit in a metal film is proposed to act as an efficient dichroic surface-plasmon-polariton (SPP) splitter, which is composed of a single nanoslit in immediate contacting with two nanogrooves with different widths. Simulations show that, due to the interferences of SPPs in the upper part of the asymmetric T-shape nanoslit, the generated SPPs propagating to the left and right directions on the front metal surface can be manipulated nearly independently by altering the right and left groove widths, respectively. Based on such effects, a dichroic SPP splitter is demonstrated and the splitting wavelengths can easily be adjusted. High splitting ratios of 31:1 and 1:12 at splitting wavelengths of 680 nm and 884 nm are numerically presented with a device's lateral dimension of only 1200 nm. Further experimental results match the simulations well.

A rapid alternative to X-ray crystallography for chiral determination: case studies of vibrational circular dichroism (VCD) to advance drug discovery projects.

The absolute stereochemistry of chiral drugs is usually established via X-ray crystallography. However, vibrational circular dichroism (VCD) spectroscopy coupled with quantum mechanics simulations offers a rapid alternative to crystallography and is readily applied to both crystalline and non-crystalline samples. VCD is an effective complement to X-ray analysis of drug candidates, and it can be used as a high-throughput means of assessing absolute stereochemistry at all phases of the discovery process (hundreds of assignments per year). The practical implementation (or fee-for-service outsourcing) of VCD and selected case studies are illustrated with an emphasis on providing utility and impact to pharmaceutical discovery programs.

PCDDB: the Protein Circular Dichroism Data Bank, a repository for circular dichroism spectral and metadata.

The Protein Circular Dichroism Data Bank (PCDDB) is a public repository that archives and freely distributes circular dichroism (CD) and synchrotron radiation CD (SRCD) spectral data and their associated experimental metadata. All entries undergo validation and curation procedures to ensure completeness, consistency and quality of the data included. A web-based interface enables users to browse and query sample types, sample conditions, experimental parameters and provides spectra in both graphical display format and as downloadable text files. The entries are linked, when appropriate, to primary sequence (UniProt) and structural (PDB) databases, as well as to secondary databases such as the Enzyme Commission functional classification database and the CATH fold classification database, as well as to literature citations. The PCDDB is available at: http://pcddb.cryst.bbk.ac.uk.

Rapid injection linear dichroism for studying the kinetics of biological processes.

Linear dichroism is defined as the differential absorbance of linearly polarized light oriented in two orthogonal directions by an aligned sample. The measurement of a linear dichroism (LD) spectrum of a sample provides two key pieces of structural information. First, that the sample and the chromophores within the sample are able to align. Second, given knowledge of the transition polarization directions of the chromophores, the orientation of the chromophores within the aligned sample can be resolved. It has been shown that LD can provide unique information on the structure of some of the more challenging biomolecular complexes. This has included macromolecular protein and peptide fibers such as actin, tubulin, and amyloids as well as protein-membrane complexes and DNA-protein complexes. Much of this work has been enabled by the development of a low volume Couette flow cell that efficiently aligns long molecules in solution. However, the current Couette system is inherently complex to assemble for each experiment and hence not suited to measurement of rapid reactions. In this paper we detail the development of the first rapid injection LD cell. The system utilizes a conventional stopped-flow injection system coupled to a modified low volume Couette cell, where a narrow bore capillary replaces the normal solid central rod. The system is shown to have similar optical characteristics to the conventional LD Couette flow cell but with the added benefit of a much shorter dead time (0.60 s compared to ~60). The rapid injection Couette cell has been used to measure the degradation of DNA by DNA exonuclease I, providing data that would not be available using a conventional system.