Circular dichroism

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Circular dichroism Bhaswati Banerjee1, Gauri Misra2 and Mohd Tashfeen Ashraf1 1

School of Biotechnology, Gautam Buddha University, Noida, India, 2Amity Institute of Biotechnology, Amity University, Noida, India

2.1 Introduction The determination of secondary and tertiary structure of biomolecules, especially proteins and nucleic acids, is integral to understanding how biomolecules acquire their native, biologically active conformation. Various techniques have been developed that enable researchers to understand how secondary and tertiary structure is formed and which noncovalent interactions are crucial in imparting structural stability. X-ray crystallography is undoubtedly one of the most sensitive techniques that elucidate the structure at atomic level; however the prerequisite of obtaining the biomolecules with high purity required for obtaining the diffraction quality crystals of biomolecules has its own limitations, which allows for the scope of other techniques to be used for studying structural details. Circular dichroism (CD) is one such technique that is routinely employed to understand the secondary as well as tertiary structure of proteins, nucleic acids, and higher structures formed by association of these molecules with their respective ligands. The ease with which a biomolecule’s structure and changes therein can be studied with CD has made it a technique of choice for researchers for solution-based studies. As with most other spectroscopic techniques, CD is also primarily used to study, in comparison to native conformation, the structural changes that accompany association/dissociation of ligands and also during unfolding/refolding of the biomolecules. Similar to other spectroscopic methods, the theoretical framework for CD is not fully developed, so the elucidation of raw data is mainly done with the help of certain empirical rules that have been devised based on the study of certain model compounds. Though there are excellent reviews on

CD, its applications [1
3], and online databases/tools/ repositories [4
11] available, in the following sections an effort has been made to put in simple terms how this technique could be used by researchers in industry and in academics to study structural changes in proteins and nucleic acids.

2.2 Principle The CD essentially deciphers the interaction of plane polarized light (PPL) with an asymmetric molecule. Unlike unpolarized light (whose E vector oscillates in all the planes), the PPL has its E vector oscillating in a single plane, which is achieved by passing unpolarized light through a polarizing material like Polaroid, nicol prism, etc. [12]. From two PPL waves of the same wavelength and amplitude that differs in phase by 1/4 of wavelength and whose E vectors are perpendicular to each other, the resultant wave’s E vector appears to oscillate in a circular fashion. This is referred to as a right circularly polarized light (CPL), if the oscillation appears to be clockwise to an observer looking at the light source while it is referred to as left CPL if the tip of resultant E vector follows anticlockwise path. If right and left CPLs of equal amplitude are superimposed the result is PPL, while if the two CPL waves are of unequal amplitude the result is elliptically polarized light. When a symmetric molecule absorbs PPL, both the right and left components are equally absorbed and the emergent light is also a PPL; however, if asymmetric/chiral molecule interacts with PPL, the right and left components of PPL will be unequally absorbed and the resultant

Data Processing Handbook for Complex Biological Data Sources. DOI: https://doi.org/10.1016/B978-0-12-816548-5.00002-2 © 2019 Elsevier Inc. All rights reserved.

21

Data Processing Handbook for Complex Biological Data Sources As mentioned earlier, certain empirical rules are generally used to interpret CD data. Following are some of the empirical rules [13]:

wave will be elliptically polarized with CD, that is, ΔƐ 5 ƐL
ƐR. Thus, a CD signal may have both negative and positive values depending on the relative absorption of right (ƐR) and left (ƐL) components. A word of caution about operation of CD instruments: the nitrogen gas should be flushed through the instrument so as to cool down the machine-lamps; this is very important when the temperature-induced transitions are studied. During such transitions, water is also continuously flushed through the instrument. The nitrogen prevents the formation of ozone caused by the lamp, which can damage the components of the CD. Fig. 2.1 gives an outline of what happens when a PPL wave interacts with a symmetric or asymmetric molecule.

1. A CD spectrum is additive; that is, it is the simple sum of the spectra of its components. This is not always strictly true but is certainly a good approximation. 2. The rotational strength (the area under the ΔƐ vs λ curve) of a CD curve is a measure of the degree of asymmetry. An agent that increases or decreases these parameters usually does so by increasing or decreasing asymmetry. Although other spectral features usually accompany the change in asymmetry. For example, a commonly used chemical agent, trifluoroethanol

z Electric field component

Electromagnetic radiation

y Magnetic field component

E vector oscillating in all planes

x Direction of propagation

Light source

Polarizing filter

Plane polarized radiation

Unpolarized radiation

E vector oscillating in a single plane Polarized light Unpolarized ray On interaction with

Asymmetric molecules

Symmetric molecules

Standard curve 80,000 CD Spectra (Unequal absorption of right and left circularly polarized light components of plane polarized radiation)

Nucleic Acid Nucleic Acid + Protein

Nucleic Acid Protein

1.0

0.6

helix sheet coils

40,000 20,000 0

0.4

0.2

0.2

Wavelength (nm)

–20,000

0.6

0.4

0.0 230 240 250 260 270 280 290

–40,000

0.0 230 240 250 260 270 280 290 Wavelength (nm)

Figure 2.1 Outcome of interaction of PPL wave with a symmetric/asymmetric molecule.

22

60,000

0.8

0.8 CD mdeg

Absorbance (Relative)

1.0

ORD Spectra (Unequal retardation of right and left circularly polarized light components of plane polarized radiation)

Ellipticity

Equal absorption of right and left circularly polarized light components of plane polarized radiation

–60,000 190

210

230

Wavelength (nm)

250

Circular dichroism

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proteins the peptide bond acts as a strong chromophore in the region 180
230 nm (far UV wavelength range) while the aromatic amino acids absorb in the range 250
300 nm (near UV wavelength range). Also, since the CD deals with differential absorption of right and left CPL components of PPL, the range in which CD signal is obtained coincides with the range of absorption. Fig. 2.2 gives CD spectra of various proteins under different conditions; the details of these are described in the legend for this figure [14,15]. Besides, Table 2.1 above gives an excellent troubleshooting guide for some of the common problems encountered while obtaining the CD data [14].

2.3 Raw data analyses Before we discuss examples to understand the analyses of raw data, certain precautions and important points need to be remembered:

Figure 2.2 Circular dichroism (CD) spectra of polypeptides and proteins with some representative secondary structures- a, CD spectra of poly-L-lysine in the 1, α-helical (black) and 2 antiparallel β-sheet (red) conformations at pH 11.1, and 3 extended conformation at pH 5.7, 3 (green) [16] and placental collagen in its 4, native triple-helical (blue) and 5, denatured (cyan) forms [17]. Note that the extended conformation of poly-L-lysine was originally described as a “random coil” but its spectrum is similar to the conformation of poly-L-proline II [18,19], which forms an extended left-handed helix.

(TFE), induces helicity in aqueous proteins; this is accompanied by an increase (more negative) in the intensity of CD signal in the far UV range, that is, 190
250 nm. 3. A chromophore that is symmetric can become asymmetric (optically active) when it is in an asymmetric environment (e.g., a helix). This may or may not be achieved by a change in λ0 (wavelength of the peak of the CD curve). 4. The value of λ0, (wavelength of maximum signal) and sign of ΔƐ at λ0 allows the chromophore to be identified because it is always very near the value of λ0 obtained from simple absorption spectroscopy. Since the CD signal deals with differential absorption of right and left CPL components of PPL, it is pertinent to mention the range of wavelength used depends on the absorption range of the chromophore. For instance, in

1. The baseline subtraction must be performed by subtracting the CD spectra of the buffer in which our molecule of interest is dissolved from the spectra obtained for compound of interest. 2. Avoid using H2O that had been stored in a polyethylene bottle for a long time. The polymer additives may elute resulting in the water losing its transparency. 3. Though the protein concentration for observing CD spectra is quite low, around 0.5 mg mL21, it is advisable to remove any chances of unforeseen aggregation by dynamic light scattering of at least by taking the absorbance spectra in wavelength range in which the protein does not absorb, that is, around 350
400 nm. 4. The difference in left and right handed absorbance A (l)
A(r) is very small (usually in the range of 0.0001) corresponding to an ellipticity of a few 1/100ths of a degree. 5. Due to high interference with solvent absorption in the UV region, only very dilute, nonabsorbing buffers are used for measurements below 200 nm.

2.3.1 Case study 1: to study acidinduced transitions in a protein Fig. 2.3A shows the far UV CD spectra of cytochrome c, a small protein of about 12,400 Daltons that contains heme prosthetic group [20]. The curve labeled 4 in Fig. 2.3A is far UV CD spectra of cytochrome c in native state while 1, 2, and 3 are acid unfolded state at pH 2.0, in the presence

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Table 2.1 Troubleshooting guide for common problems encountered in CD and their solutions. S. No. Problem

Solution

1.

The CD spectra have very low ellipticity.

Check the protein concentration.

2.

The signal to noise is very low.

Try opening the slit width to 2 nm, as the exact wavelength is not critical. Try increasing the time of data collection. Signal to noise increases as the square root of the time of the signal averaging time.

3.

The samples precipitate when heated.

Try using a different buffer or include stabilizing agents such as low concentrations of glycerol.

4.

The samples refold to give the same spectra as the starting material but the unfolding and refolding curves are displaced from each other.

The samples are not at thermal equilibrium during the measurements. Try increasing the equilibration time.

5.

The curve of ellipticity as a function of temperature appears to have a typical sigmoidal shape, but none of the folding equations fit the data and the error of the fit is large.

Make sure that the initial parameters are close to the actual values of the data and that the correct units are used for the initial estimates of the ellipticity of the folded and unfolded protein (e.g., millidegrees or mean residue ellipticity). Make sure the initial estimate of the TM of folding is close to the midpoint of the transition. If this doesn’t work, try increasing or decreasing the initial estimates of the enthalpy of folding.

6.

The calculated TM values for different concentrations of protein are not close to each other, even when the data are modeled to fit the dissociation of dimers or trimers.

Determine the oligomerization state of the protein using independent methods such as gel filtration or ultracentrifugation, and use the van’t Hoff equation to determine the thermodynamic parameters.

7.

The macro doesn’t work properly.

Check the users manual to make sure you have programmed the machine properly. Usually machines will have sample macros. Try to program a single temperature step with using only one or two samples.

8.

A. When the spectra are deconvoluted using the CCA algorithm some basis curves only contribute to a spectrum obtained at a single temperature. B. When the spectra are analyzed using the CCA algorithm, some of the curves have very similar shapes but are displaced from each other.

A. If a spectrum is noisy and has outlying points, the CCA algorithm will identify the spectrum as a unique basis set. Try removing the spectrum from the data set. B. The CD spectra of the cells may change as a function of temperature, leading to shifts in the baseline. Obtain spectra of the cuvettes filled with water as a function of temperature and correct the individual data set for the contribution of the cells. C. If the spectra of the folded and unfolded proteins have maxima (nodes) that are identical to each other, SVD the solution may be “singular” and only one spectrum will deconvoluted.

C. When the data is deconvoluted using singular value decomposition (SVD), while it is clear there are more than two states, only one principal component is resolved.

Source: Adapted from Greenfield NJ. Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat Prot 2006;1(6):2527
35.

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Circular dichroism

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MRE × 10–3, deg.cm2.dmol–1

(A)

0 1 2 3 4 –10 200

210

220

230

240

250

(B) 4

MRE, deg.cm2.dmol–1

290

2 1 3

0

–150 250

260

270

280

290

300

Wavelength (nm)

Figure 2.3 Cyt c far-UV (A) and near UV CD spectra (B): (1) acid unfolded state at pH 2.0, 10 mM glycine HCl buffer; (2) 33 mM TCA-induced state; (3) 3.3 mM TCA-induced state; (4) native state at pH 7.0, 10 mM sodium phosphate buffer. Protein concentration was 26 (A) and 50 μM (B). The path length was 0.1 (A) and 1 cm (B).

of 33 mM TCA and in the presence of 3.3 mM TCA, respectively. Since cytochrome c is an α-rich protein, two shoulders are observed in far UV CD spectra, at 208 and 222 nm, which is a characteristic feature of proteins that have a significant amount of α-rich regions. It can be seen that the intensity of signal in all three unfolded states is far less as compared with the native state. Note that the far UV CD spectrum gives details about the secondary structure of biomolecules (i.e., primarily stabilized by hydrogen-bonding interactions between amino acids that lie close together in primary sequence). As mentioned above, the main chromophore in this region is the amide linkage of peptide bond. On the other hand, the near UV CD spectrum gives a rough idea of the overall threedimensional structure of proteins; the main chromophores in this region being the aromatic amino acids, that is, tryptophan, tyrosine, and to some extent phenylalanine. Fig. 2.3B above shows the near UV CD spectrum of cytochrome c under conditions similar to Fig. 2.3A. As near UV CD spectra gives the global picture, it is difficult to deduce the contribution/influence of specific aromatic amino acid. Therefore, the near UV CD spectra are generally used to observe the change in the spectrum between

different experimental conditions. Far UV CD spectra, in contrast, may be used for more quantitative information, in terms of percentage of residues involved in forming the helix. The α-helical content of a protein can be calculated from the MRE value at 222 nm using the following equation as described by Chen et al. [21]: % α 2 helix 5

MRE222
2340 30; 300 3 100

where MRE is mean residue ellipiticity; MRE222 is mean residue ellipticity at 222 nm.

2.3.2 Case study 2: to study pHinduced transitions in a protein In one of the studies conducted to study the effect of alkaline pH on Concanavalin A (Con A) in the presence/ absence of metal ion (Mg21) [20], certain interesting results were obtained as shown in Fig. 2.4. It shows the far-UV CD spectra of Con A under different pH conditions between pH 7 and 12. The spectrum of Con A at pH 12 shows the structural transition with a CD band at 212 nm (Curve 5), a shift

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Data Processing Handbook for Complex Biological Data Sources

MRE × 10–3 deg.cm2.dmol–1

6.75

0 6 1 2 3

–4.5 5 –9.0 200

4 210

230 220 Wavelength (nm)

240

250

Figure 2.4 Far-UV CD spectra of Con A at pH 7 (Curve 1); pH 7, metallized (Curve 2); pH 7 demetallized (Curve 3); pH 12 (Curve 5); pH 12, demetallized (Curve 4) and in the presence of 6 M Gdn HCl (Curve 6).

of around 11 nm from that of 223 nm for native Con A at pH 7 (Curve 1). The signal at 217 nm at pH 12 was also considerably enhanced as compared with that at pH 7. Though the presence of metal ion/EGTA at pH 7 was not found to significantly affect the signal in the far-UV region (Curve 1 and 2 respectively) but the presence of metal ions at pH 12 was found to aggregate the protein. On the other hand, the presence of EGTA at pH 12 (Curve 4) showed similar transition as that of apoprotein at pH 12 (Curve 5) but with enhanced signal intensity. Curve 6 shows the far UV CD spectra of Con A denatured in the presence of 6 M Guanidine hydrochloride (Gdn HCl).

2.3.3 Case study 3: to study structural transitions in nucleic acids The structural changes in nucleic acids, DNA and RNA, can also be studied using CD spectroscopy. Though the nitrogenous bases per se are not chiral but the chirality gets induced due to nearby chiral sugar present in ribo/ deoxyribonucleotides. This induction of chirality is illustrated in Fig. 2.5 below. In DNA, the helical structure can be experimentally determined by CD spectra. The sign and shape of the CD spectra are different for B-DNA, which has a right-handed double-helical structure as compared with the Z-DNA, which has left-handed double-helical structure. In

26

Figure 2.5 Circular dichroism of DNA, RNA, and free ribo/ deoxyribonucleotides.

particular, for B-DNA the signal at around 295 nm in CD spectra is positive while in the case of Z-DNA it is just the opposite. Following are three important factors that affect the CD spectra of DNA: the conformation of dG monomer, the hydrogen-bonding interaction between two helices, and lastly, the stacking interactions between nucleic acid bases.

2.3.4 Case study 4: determination of Tm and other thermodynamic parameters CD is one of the simplest spectroscopic techniques used for measuring the thermal stability of proteins in natively

Circular dichroism folded and different partially folded/unfolded conformations. Using CD, the fraction of molecule that is in native/ denatured state under given conditions can be calculated. For this, the MRE values at particular wavelength (e.g., at 222 nm for an alpha-rich protein) are used to calculate fraction denatured (fD), which is then plotted against temperature/pH/denaturant concentrations. Assuming two-state protein folding, denaturation midpoint is defined as that temperature (Tm) or denaturant concentration (Cm) at which both the folded and unfolded states are equally populated at equilibrium. Tm is often determined using a thermal shift assay. For calculating melting temperature of a protein, let’s consider a protein and a single point mutation in two states, native (folded) and denatured (unfolded) [22]. The equilibrium is defined like any other reaction: Keq 5 [denatured protein]/[native protein]. If one uses PPL or fluorescence to determine the fraction of a protein’s folded or unfolded conformations at different temperatures, a protein-melting curve can be produced [23]. To determine the folded fraction from the thermal denaturation experiments, the following formula can be used [23]: ½θobs
½θden ½θnat
½θden where [θ]obs is the ellipticity at a given temperature, [θ]den represents the ellipiticity at highest temperature and [θ]nat at lowest temperature, respectively. An extensively studied example for the usage of CD in understanding the thermal stability profile of a chaperone is Pf Hsp70-1 from Plasmodium falciparum and its truncated variants. The main transition is observed in the range of 30
45 C, thereafter loss of intensity at θ222 nm followed by a second transition at 80 C (Fig. 2.6A). A single change in thermal denaturation curve was observed with Tm value of 45 C for its nucleotide binding domain (NBD), pointing towards cooperative unfolding with no intermediate stages and following the standard two-state model of unfolding (Fig. 2.6B). Its other domain, namely substrate binding domain (SBD), shows transition at approximately 40 C, retaining 80% of the compact structure (Fig. 2.6C). SBD combined with the C-terminal domain of this protein exhibits folded structure till 80 C, which later causes a decline in θ222 nm intensity (Fig. 2.6D). These results have remarkably established that the structural stability of the PfHsp70-1 is contributed majorly by the C-terminal domain in complex with the SBD as the latter truncated mutants enhanced the stability of the otherwise unstable NBD, reflected from CD studies (Fig. 2.6E). CD is also used to study the structural changes induced in Pf Hsp70-1 on chemical denaturation using urea

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(Fig. 2.7) and GdnCl (Fig. 2.8) by monitoring the changes in the ellipticity at θ222 nm in the presence of increasing concentrations of respective denaturants [23]. The above examples clearly demonstrate the application of CD for the determination of structural changes and stability in response to the changes in the immediate environment. Such applications are extensively useful for gaining structural and functional insights of proteins. Both the enthalpy (H) and entropy (S) can be calculated using a curve of a fraction of protein in folded and unfolded state. The S and H values can be calculated using the van’t Hoff equation and plot. The van’t Hoff equation explains the temperature dependence of the equilibrium constant. To use this equation, the protein must refold—this is an equilibrium problem! The van’t Hoff equation is derived from Gibb’s free energy equation: ΔG 5 ΔH
T ΔS With the understanding that for this reaction of folding and unfolding: G 5 2RT lnKeq, one can create the van’t Hoff equation to relate equilibrium constant to temperature by substituting the two equations and rearranging them to generate the van’t Hoff equation: lnKeq 5

R

2H

1 RS

1 T

Since the equation is a straight line equation, the plot of lnKeq versus 1/T, known as the van’t Hoff plot, yields a straight line with slope
H/RT and intercept on Yaxis 5 S/R. Using both the melting or transition curve and van’t Hoff’s plot and equation, one can determine the thermodynamic functions of protein stability (the fraction of protein at a given temperature that is native or denatured). The van’t Hoff plot helps determine the fraction of protein folded from the transition curve where [native protein] 5 [denatured protein] and convert that information to Keq for each temperature. From this data one creates a van’t Hoff plot and calculates the enthalpy and entropy from the slope and Y intercept. To determine the stability of a protein accompanying a change (ligand binding, protein interaction or mutation), one needs to determine the ΔG for each conformational transition, for instance in the case of development of mutant, ΔG (protein) 5 G wild-type
G mutant. A positive value gives clear indication that the unfolding of wild type protein is less favorable than the mutant as per the calculated value, thereby meaning that the mutation decreases the stability of the native protein. On the other hand, a negative value indicates the unfolding of the wild type is more favorable than the mutant or that the mutation stabilizes the native structure.

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Data Processing Handbook for Complex Biological Data Sources

Figure 2.6 Circular dichroism-based thermal stability analysis of native and truncated mutants of (A) PfHsp70-1, (B) NBD, (C) SBD, and (D) SBD along with C-terminal tail. Inset depicts far-UV spectra for each of the constructs before (bHS) and after (aHS) heat denaturation. (E) Folded fraction of NBD, SBD 1 NBD and SBD with C-terminal 1 NBD in equimolar ratio as measured by changes in CD ellipticity at 222 nm. Adapted from Misra G, Ramachandran R. Hsp70-1 from Plasmodium falciparum: Protein stability, domain analysis and chaperone activity. Biophys Chem 2009;142:55
64.

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Circular dichroism

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64% of its residues are engaged in forming β-pleated sheets. The native conformation of Con A is a tetramer that is composed of a dimer of two dimers. The tetramer has a characteristic trough around 223 nm in far UV CD region; any substantial change in secondary structure of Con A is accompanied by a change in CD signal at 223 nm. Thus, the changes in secondary structure of both α- and β-rich proteins can be studied by far UV CD spectroscopy. The effect of certain helix-inducing agents like TFE and HFIP (hexefluoroisopropanol) can be studied using the far UV CD spectroscopy. Apart from solvent perturbation studies, the change in structure on binding of ligand (e.g., drug molecule, like an enzyme inhibitor) to a protein can be studied by CD spectroscopy. Figure 2.7 Changes in PfHsp70-1 θ222 with increase in urea concentration. Adapted from Misra G, Ramachandran R. Hsp70-1 from Plasmodium falciparum: Protein stability, domain analysis and chaperone activity. Biophys Chem 2009;142:55
64.

Figure 2.8 Changes in PfHsp70-1 θ222 with increase in GdnCl concentration. Adapted from Misra G, Ramachandran R. Hsp70-1 from Plasmodium falciparum: Protein stability, domain analysis and chaperone activity. Biophys Chem 2009;142:55
64.

2.4 Miscellaneous examples Since the β-rich aqueous proteins have their characteristic far UV CD spectrum, the changes in their structure can be easily studied by CD. For instance, Con A is an extensively studied lectin that is rich in β-rich regions; around

2.5 Conclusion CD is an important noninvasive spectroscopic technique for understanding the structure of biomolecules in solution and therefore plays an important role in establishing the structure
function relationship in biomolecules like proteins. Relative changes in structure due to influence of environment on sample (pH, denaturants, temperature, etc.) can be monitored very accurately. In particular, the fine details of secondary structure as well as changes in the tertiary structure of biomolecules like proteins can be studied. For α-helical rich proteins the number of residues involved in helix formation can be determined. Though the application of near UV CD spectroscopy has not been that instrumental, it is being successfully used to monitor the changes in the overall three-dimensional structure of protein.

Acknowledgments The authors have no conflict of interest. The authors are thankful to Priyansh Srivastava, Fauzan Ahmed from Amity Institute of Biotechnology, Amity University, Noida and Srishti Jha, School of Biotechnology, Gautam Buddha University for their valuable contribution in preparation of this work, especially the illustrations, which convey points that would otherwise be difficult to put forth in words. The authors are also thankful to Gautam Buddha University for providing the required infrastructure.

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