Circular Dichroism, Applications

Giuliano Siligardi and Rohanah Hussain, Diamond Light Source Ltd., Diamond House, Didcot, Oxfordshire, UK
ã 2017 Elsevier Ltd. All rights reserved.

Symbols [L] concentration of a bound ligand
DA differential absorbance [HL] concentration of a bound host–ligand complex
AL absorbance from left-circularly polarized light K equilibrium binding constant
AR absorbance from right-circularly polarized light D« differential molar extinction coefficient
[HT] total concentration of a host « molar extinction coefficient
[LT] total concentration of a ligand c concentration in mol l1
[H] concentration of a bound host l cuvette pathlength in cm

Abbreviations OR optical rotation
CD Circular dichroism (CD) ORD optical rotatory dispersion
ECD electronic CD ROA Raman optical activity
HL host–ligand complex VCD vibrational CD
ITC isothermal titration calorimetry

Introduction spectra is less user-friendly than the other techniques. The
analysis of the data is not yet available as a comprehensive
The most important and frequently used techniques to study, software package like for ITC.
monitor, characterize, and analyze binding interactions of The ‘golden’ rules on how to carry out good ligand titrations
nonimmobilized molecules in solution are isothermal titration and what protein–ligand concentrations to choose in order to
calorimetry (ITC), fluorescence, and circular dichroism (CD). study binding interactions of biologically important molecules
For a detailed and thorough study of complex biosystems, one by CD spectroscopy are described in here.
technique is seldom enough. This is also because each tech-
nique has its own limitations.
ITC requires a significant amount of sample, particularly, Optical Activity
when the heat released upon binding is rather small. For more
complex systems where multiequilibria may exist, for instance, A chiral molecule is not superimposable on its mirror image
intra- and intermolecular interactions (association), the bind- and as a consequence it shows optical activity in the form of
ing can be very difficult to be determined correctly particularly optical rotation (OR), optical rotatory dispersion (ORD), CD,
when the ligand and the host are in different solvents due to and Raman optical activity (ROA).
solubility problems. Linearly polarized light can be seen as the sum of two
Fluorescence relies on the presence of fluorophores with circularly polarized lights in opposite directions, one left and
sufficient intrinsic fluorescence. The majority of proteins con- one right.
tain useful fluorophores such as tryptophan and tyrosine OR is the difference in speed between the propagation of
amino acid residues that can be used to probe binding inter- left- and right-circularly polarized lights that pass through a
actions. However, their intrinsic fluorescence is rather modest. chiral medium. With no light absorption from the sample, the
The introduction of more sensitive fluorophore probes by result is the rotation of the plane of linearly polarized light.
chemical tagging might induce positive or negative results by ORD is the change of OR as a function of wavelength.
promoting or hindering the binding interactions. This is CD is a small difference in the absorption between left- and
encountered in high-throughput drug screening where the reli- right-circularly polarized lights of a chiral molecule. Electronic
ability of positive and negative results to select lead com- CD (ECD) is generated by molecules with chromophores
pounds cannot be dismissed. The open question is how good absorbing in the UV and visible spectral regions, whereas
is the screening test, in other words how many positive results vibrational CD (VCD) is generated in the IR spectral region.
are biased by the artificial binding promoted by the tagged ROA is a small difference in Raman intensity between right-
fluorophore and how many bindings are missed, that is, neg- and left-circularly polarized lights or scattered circularly polar-
ative results, induced by the steric hindrance of the tag moiety? ized light of a chiral molecule.
CD spectroscopy has none of these limitations; however, Both CD and ROA arise from the absolute configuration
the setting of the optimal instrumental and experimental (the chirality, or handedness) and the molecular structure (the
parameters to obtain the necessary good signal-to-noise CD conformation).

Encyclopedia of Spectroscopy and Spectrometry, Third Edition 293

5 nucleic acids. made of L-amino acid residues. The quadratic equation to calculate Kd has been derived Rather demanding calculations including solvent interactions from a two-component system where only one component for larger and more flexible molecules (peptides) with about has intrinsic CD (Figure 1). maybe in the next decade thanks to less computationally expensive calculations. The local tertiary structure of the aromatic amino acid residues 0 observed in the near-UV region (250–330 nm) can be used for quality control as it often reveals subtle changes from batch to P3 batch often not mirrored in the far-UV region. Figure 1 CD spectra of the titration of proline-rich peptides P2 (primary whereas in the 250–350 nm region the absorption in a 1 cm sequence PPPLPPKPKF) and P3 (PPPNSPRPGPPP) into aqueous GST- pathlength is approximately 1–1. To add 1 molar equivalent SH3 protein domain (60 mM). and ROA spectra from a 1:6 pool of conformations sampled from quantum mechanical 1:3 molecular dynamics. In the 280–330 nm region.6 the technique of choice to investigate and characterize struc.0 E−4 [HT]¼[H]þ[HL]. and Beer’s law A¼e c l with l¼1 cm: DA ¼ ðDeH  DeH Þ ðK½HT  þ K½LT  þ 1Þ  ððK½HT  þ K½LT  þ 1Þ2  4KK½HT ½LT Þ2  −2. the concentration of the stock cules with up to 30 atoms can now be determined. The main task of this review is to facilitate and improve this diffusion.05 cm pathlength is approximately 0. −2. [LT]¼[L]þ[HL].5 E−4 2K þ DeH ½HT  P3 (Kd = >2 mM) [1] Usually CD titration is conducted in a stepwise manner. The 0 implication is that the 3D structure of proteins in solution P2 could be determined one day with sufficient resolution from observed and calculated ECD. thanks to solution to be prepared is approximately 50-fold that of the the revolutionary developments of density functional theory concentration of the host component. relatively rigid mole. and estimate the pro- tein secondary structure in the far-UV region (180–250 nm). 1:0. methods for the calculation of ECD.3 tural molecular behavior in solution. CD is advan- tageously used to monitor.8. eqn [1] a not distant future.4 μM) sis.294 Circular Dichroism. when both host and 120 atoms have been recently achieved. such as proteins. characterize. 1:2 For biologically important molecules. of ligand in a 20 ml volume. both peptides . carbohydrates. It is conceivable that in ligand components possess intrinsic CD (Figure 2). and lipids. adding small aliquots of 3–5 ml of ligand stock solution directly −3. VCD. Applications The absolute configuration of small. The application of CD spectroscopy to determine binding −10 E−5 interactions is not as diffused as it should be. VCD.4. for a single binding site. 1:1. ΔA = (AL−AR) Another important use of CD is to probe qualitatively and −5E−5 quantitatively binding interactions in a solution of biomole- cules without the need of immobilizing and labeling them. 1:0 For proteins. the apparent dissoci- ation constant Kd¼1/K is determined by analyzing the CD data ΔA = (AL−AR) (291 nm) expressed in DA¼(ALAR) using a nonlinear regression analy- P2 (K d = 1. However.5 E−4 between the host (H) and the ligand (L). small proteins will be tackled leading the way to even larger protein systems. Binding Interactions of Nonimmobilized and 300 320 Nonlabeled Biomolecules in Solution by CD Wavelength (nm) Spectroscopy For a host–ligand (HL) complex formed from the interaction −1. and ROA spectra.0 E−4 into a cuvette of appropriate pathlength that contains a known 0 100 200 300 volume of the host component to be titrated. For measure- Proline-rich peptide (μM) ments in the 190–250 nm spectral region the optimum UV absorption in a 0. CD spectroscopy is 1:1 1:0. At equilibrium. eqn [1] used to determine the Kd has been derived from DA¼DAHLþDAHþDAL.

As both proteins have plenty of aromatic residues.4 0. above all. Circular Dichroism. parameters to obtain the best signal-to-noise ratio spectra. nonlinear regression analysis from the software program Origin. a 2–3 molar excess of ligand is required for figure as well as in the other figures.4 1:0.8 1:0. the stoichiometry of the binding.0 [p50] 0.1 1:0. Germany) with a volume of for the binding interaction study.8 1.0 0. In this from both proteins.4 1:1.0005 1:1.8 0 0 260 280 300 Wavelength (nm) 260 280 300 ΔA = (AL−AR) (293 nm) 1:1 ΔA = (AL−AR) (293 nm) Kd = 2.0010 A:B A:B 1:2. whereas.5 µM 0. Müllheim. To obtain high accuracy.6 0. GST-SH3 difference CD spectra. accurate results. Using different plotting the intensity of differential CD or CD at single wave- instruments and of different brands. to be used 1 cm cylindrical cell (Hellma. This can be obtained by subtracting domain shows a CD signal that is changing upon addition of P2 but not from each observed CD spectrum of the HL (1:n) mixture the for P3. Multiscan mode was used to improve the signal-to-noise ratio. The 1:1 stoichiometry of the titration was revealed by manner the plot DA versus ligand concentration reaches saturation.2 0. CD spectra were measured using a stronger bindings. A special The assessment of the experimental parameters. it is important to find the optimum length versus ligand molar fraction (Figures 2 and 4). and bandwidth of 2 nm were used.0 1:2. With the J720 a scan speed of 20 nm min1.1 ΔA = (AL−AR) 0. The concentration was determined by weighing using a microbalance with 1 mg sensitivity (Mettler Toledo. it is important to terminate plot DA intensity at 291 nm versus peptide concentration using a the titration upon reaching saturation or as close as possible.1 1:0. a tight is achieved because any inaccuracy will be reflected in the determination binding affinity with Kd in the 10–20 nM range requires a of the Kd as well. 4–6 molar excess or even higher. the plot DA intensity at 293 nm versus molar fraction of [p50]/{[p50]þ otherwise it will look like the stoichiometry plot of Figure 2. can be converted back into one active component system using have no CD due to the lack of Trp and Tyr aromatic residues. In this As a rule of thumb.0 Figure 3 Difference CD spectra of the titration of p50 protein into hsp90 [p50]/{[hsp90]+[p50]} protein. The determination of accurate protein concentration is Figure 1. the concentration is approximately 50–100-fold that not trivial.8 1:1. Special care should be taken to ensure that good accuracy of the dissociation constant Kd¼1/K. system to system but either four or nine scans with the highest scan Important information that can be obtained with the Kd is speed are normally used. The Kd was determined by fitting the (Figure 3).4 1:0. host solution of approximately 5–10 mM concentration . is essential to obtain mean- 520 ml was used to carry out the CD titrations. This is unambiguously indicative of binding interactions between equivalent CD contribution of the ligand of n molar ratio GST-SH3 domain and P2 peptide. For example.0 1:2.8 1:0. Applications 295 0. OH).0 0. is Jasco J720 spectropolarimeter purged with nitrogen gas. This varies from needed for weaker bindings. a Michaelis–Menten type of saturation curve illustrated in Columbus.4 ΔA = (AL−AR) 1:1. the CD arises different molar ratios the equivalent CD contribution of p50 alone.1 1:2. This can be calculated by time constant of 4 s. of 2–20 and 20–200 ml tips (Finnpipette) were used to directly add What is the right concentration for the host component aliquots of ligand protein or peptide to the host protein placed in the to be titrated by the ligand? To obtain a curve fitting like cuvette cell of suprasil material. As both proteins contribute to the CD spectrum. Positive display pipettes ingful and. [hsp90}. the Kd is Figure 2 CD spectra of the titration of p50 protein into hsp90 protein determined by subtracting from the CD spectra of hsp90 with p50 at (96 mM).

69 1:1.0 Figure 5 CD titration of a sugar ligand into lysozyme at different 1:2 concentrations. For instance. The discrimination between 1 and sent in the titration can be inferred when. the range of one or more. a very sharp edge cannot be discriminated very well whereas Indications that more than one equilibrium might be pre- the more parabolic one can.12 1:0.06 15 1:3 1:0. The sugar moiety is transparent in the near-UV region.99 factor is used to determine the cell pathlength. Applications 1:1. parabolic shape (Figure 5). requires a host solution of 50–100 mM concentration. eqn [1] very well fits binding interactions of one single binding site. With lower host concentration the curve becomes parabolic and higher ligand excess is required to accurately fit the binding curve. there is no need to transform the CD data into different CD data like for Figures 2 and 3. at high ligand molar 10 nM Kd curves (Figure 6) can be achieved by repeating the excesses. dilution means that to measure a good signal-to-noise spec- trum a longer pathlength has to be used.33 0.5 µM) 6 5 Kd = 1. the fitting curve is of equivalent ligand concentration. The 2:1 stoichiometry components is nevertheless revealing.16 µM µ 7 Lyz (17. The scattering at longer wavelengths is observed.0 4 Sba1 (M) 0 2 4 6 0. can be verified by an accurate determination of the Kd of binding interactions that can be successfully deter.78 1:0. ciation might obscure the binding site.66 0. Obviously for more than In Figure 6 are illustrated several simulated fitting curves one binding site. Ideally the dilution 0. If the Only the fraction of available binding site can interact with the host concentration is less than 100 Kd. The fitted curves showing mine the Kd. the host and ligand concentrations.00 1:0.00 0. therefore. The host or ligand . In general.3 µM 0.50 ΔA = (AL−AR) (10−5) 1:2 1:0. the experimental CD data cannot be fitted and light titration with a more dilute total host concentration. the stoichiometry. Figure 4 Difference CD spectra of the titration of Sba1 into preformed Failure to fit the experimental data due to wrong concentration hsp90þAMP–PNP (1:2) complex (30 mM).25 10 1:1 1:0. eqn [1] cannot be used qualitatively to deter- using Kd values from 1 nM to 10 mM. By CD spectroscopy. the Kd remains the same but the fitting is no longer of Michaelis–Menten type using the highest host concentration. host asso- indicates that one molecule of p23 protein binds to an hsp90 dimer. which requires an accurate determination of mined spans from millimolar to nanomolar. As the affinity is concentration independent. The number of binding sites.296 Circular Dichroism.38 1:0. In this case there is a struggle to fit the slope of the first experimental data points reaching 1:1 molar ratio and the data can be fitted only by whereas a medium affinity with Kd in the 1–10 mM range reducing the total host concentration in the regression analysis.4 0 5 1:0 0 280 300 320 260 280 300 9 ΔA = (AL−AR) (10−5) λ = 294 nm 8 Lyz (445 µM) K d = 1.

2. 2nd edn Cambridge University Press. 0. Chiroptical Sensors. 0.001. Theory. Instrumentation. the total concentration reached upon saturation 10 µM cannot exceed UV absorption of 0. The first is the formation of the Chemistry Applications. The Spectrometry. Linear Dichroism. Vibrational CD Hsp90–p50 complex followed by p50 association for greater 1:1 molar Spectrometers.2–1. 1.0 of UV cuvette cells of different pathlength (0. Biomacromolecular Applications. Exciton Coupling. From Beer’s law. and Siligardi G (1998) Marine metabolites and might precipitate once saturation is reached.0 residues. Perkin Transactions 2: 129–135. 0. accurately as possible the binding affinity of molecular interactions. Raman Optical Activity. the molar extinction coefficient can be approxi- mately calculated from the number of Trp. Stereochemistry Studied Using Mass Figure 7 Analysis of Hsp90–p50 interactions by near-UV CD. UV- ligand p50 has a weaker tendency to interact with itself than to Hsp90. plotted curves change in terms of profile. Kd = 2. The cyclopeptides. For proteins and peptides. spectra for b-hairpin peptides stabilized by an Aib-Gly turn sequence: Correlation ditions to solve or improve the solubility issues hence allowing between conformational fluctuation and vibrational coupling. Chiroptical Spectroscopy. increased photon flux of the light source been calculated with different Kd: 1 nM. the absorption. assuming Determination of Absolute Configuration. Ligand (M) 005. Induced. Inorganic hence. Circular Dichroism. Raman Optical Activity. Chiroptical Spectroscopy. Circularly Polarized Luminescence and Fluorescence Detected Circular Dichroism.5 mM. Bour P. Visible Absorption and Circular Dichroism Spectroscopy. it will give an apparent Kd of 2. In this example the initial total For small intrinsic CD signals. Drake AF. Protein Structure Analysis by CD. Linear Dichroism. components might be unstable in their unbound forms and Freeman DJ. General Theory. 2 mM. and S–S bonds present in the primary sequence. Theory. 1 mM. . and Keiderling TA (2006) Similation of infrared titration might be repeated in different buffer or solvent con. The fitted curves tions. Theory and Application to excess concentration. 10 nM. Journal of Chemical Society. For protein–protein or protein–peptide titrations. 8 E04 1:1 See also: Absolute Stereochemistry by NMR Spectroscopy. Oriented Molecules and Anisotropic Systems. the choice of instrumental host concentration was 10 mM. Ellipsometry. Surface-Enhanced Raman Optical Activity (SEROA). Circular dichroism studies of metal binding to Lissoclinum this case is the clear lack of saturation plateau (Figure 7).4 in the 250–340 nm spectral region (aromatic residues and disulfide bond) otherwise signal cutoff is reached. Journal of Molecular Structure (THEOCHEM) 357: 225–235. 100 nM. 0. 0. Applications. the titration will show two events. and 10 mM. the calculation of the cell path- length requires the knowledge of the concentration. Applications. Small 0 Molecule Applications. Chiroptical Spectroscopy. Applications 297 1 nM Once the host concentration has been chosen. Vibrational CD. Raman Optical Activity. Physical Chemistry B 110: 23590–23602. Vibrational CD. multi- experimental CD data point at 286 nm while the simulated CD curves have scan acquisition. needs to be optimized for each titration in order to improve 5 mM. in order to obtain CD measurements with the highest have been simulated using different values of Kd¼1/K to show how the signal-to-noise ratio. The Journal of a more accurate Kd determination. Kim J. Huang R. Phe amino acid 0. longer for dilute and shorter for concentrated solu- Figure 6 CD spectra calculated using eqn [1] of the nonlinear regression analysis with in the software program Origin.5.01. Raman hP50 (M) Optical Activity.02. and 5 cm) will allow the right choice of pathlength. The signature for metal ion chelation. The fitting can be still carried out and. Barron L (2004) Molecular Light Scattering and Optical Activity.1. Patteenden G. and the molar extinction coefficient. A set 0. Tyr.5 µM Circular Dichroism Spectrometers. Finley JW and Stephens PJ (1995) Density functional theory calculations of molecular structures and harmonic vibrational frequencies using hybrid density functionals. Circular Dichroism and 6 E04 Hsp90 + hP50 ORD. Emission Theory. Vibrational CD. Kubelka J. The black solid squares are the parameters such as integration time or time constant. Theory. Applications. Magnetic 4 E04 Circular Dichroism. Macromolecule and Biological Molecule Applications.8 in the 190–260 nm (back- bone region) and 1. the plateau occurred at approximately 1:1 molar ratio concentration. Note that the simulated CD spectra using 1 nM and the signal-to-noise ratio that is paramount to determine as 10 nM Kd cannot be discriminated as they superimposed one another. FTIR. Raman Optical Activity. Enantiomeric Purity Studied Using NMR. the choice of the pathlength is dictated by the molar extinction coefficient of the chromophore. and Raman Spectroscopies.2. It is important to note that other spectroscopic techniques will only at best give the overall picture of both Further Reading events without allowing an educated guess of the strengths of the interactions. ORD and Polarimetry Instruments. Spectrometers. Circular Dichroism.

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