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The Principles and Biological Applications

Of Circular Dichorism

Kamalpreet Singh

Abstract

The rapid growth in the field of biochemistry has led to an increasing demand for efficient

structural techniques. Here, a commonly employed structural technique, circular dichroism (CD)

spectroscopy, is presented. This technique, employs spectropolarimeters, which utilize the

differential absorption of left and right circularly polarized light by chiral chromophores. It can

be used in the study of various biomolecules including proteins, providing information regarding

structure and structural changes with varying conditions. Relative to NMR and X-ray

approaches, CD offers poor resolution, but greater sensitivity and efficiency, requiring less time

and a smaller sample.

The rapid growth in structural

techniques of biochemistry has allowed for the

development of numerous de novo

biomolecules [1]. This is especially apparent in

protein projects where structures of natural

motifs act as exceptional templates for design.

Examples of projects that have utilized

structural techniques in the study of proteins,

include the construction of focused protein

libraries by Hecht and associates [2], and the

production of a 23 residue protein by Barbara

Imperiali et. al [3]. Evidently, structural

techniques have become highly valuable tools

in the world of biochemistry. This article aims

to provide an introduction to the principles of a

commonly employed structural technique,

Circular Dichorism (CD) spectroscopy, and

discuss some biological applications.

Principles of CD Spectroscopy

CD spectroscopy, much like other

spectroscopy techniques, works via exploitation

of light properties. So in order to truly

understand how this technique works, one must

first understand some light properties.

Light is polarized

Invisible to the human eye, all sources

of illumination produce light with electric field

vectors that vibrate in planes perpendicular to

the direction of propagation[4] [5]. If these

vectors are restricted to a single plane, the light

is said to be linearly polarized [4] [5] [6]. If the

vectors vibrate in multiple planes with respect

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to propagation such that all vibrations cancel,

the light is said to be non-polarized [5] [6]. This

phenomenon is shown in figure 1. Note that

these two modes of polarization represent

extremes of a spectrum. In other words, there

exists several other states of polarized light in

which the electric field vectors are oriented

such that an ellipse shape is forged around the

direction of propagation [4]. Light waves

undergoing such polarization are said to be

elliptically polarized (figure2) [4] [5]. In certain

cases, the major and minor axes of the vectors

in the ellipse may be equal, leading to a circular

shape. Light waves undergoing such

polarization are said to be circularly polarized

(figure2). [4][5][6]

CD is differential absorption of L/R polarized

light

As shown in figure two, circularly

polarized light contains a rotational element.

Inspection of this rotational element from an

end on view (figure 3) [1] demonstrates that the

polarization may be left-handed or right-handed

[4]. The left and right handed polarized lights

can be thought of as enantiomers, carrying

identical properties except that they interact

with asymmetric molecules in different ways [9].

In particular, chiral objects absorb the left and

right circularly polarized light to different

extents, resulting in some net elliptically

polarized radiation [1] [10]. This phenomenon of

differential absorption is referred to as Circular

Dichorism (CD) [1] [5] [10]. It can be

mathematically represented by[10] : ∆𝐴 = 𝐴𝐿 −

𝐴𝑅 . Where ∆𝐴 represents the difference in

absorption of left and right circularly polarized

light (circular dichroism), AL represents the

absorption of left circularly polarized light and

AR represnts the absorption of right circularly

polarized light.

Figure 1. Light consist of electric field vectors that

vibrate in planes perpendicular to the direction of

propagation. Light with vectors restricted to one

plane is linearly polarized (left), while light

consisting of vectors oriented randomly with

respect to propagation is non-polarized (right). This

image was adapted from reference [7].

Figure 2. A representation of the three different

types of light polarizations discussed. This image

was obtained from reference [8].

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CD active molecules have chromophores

In order for a molecule to exhibit a CD

signal, it must contain a chiral chromophore (an

asymmetric species that absorbs light at some

characteristic wavelength) [1]. This requirement

for chirality of the chromophore is achieved in

three ways. The chromophore may be

inherently chiral due to its structure, it may be

linked to a chiral centre in the molecule, or it

may be located in an asymmetric environment

within the 3-D shape adopted by the molecule

[1]. For a more complete treatment of the

theoretical aspects of CD, consult references

[1], [5], [10-14].

Figure 3. Circularly polarized light maybe right or

left handed. Differential absorbance of L/R

polarized light (Circular Dichroism) leads to a net

elliptically polarized radiation. This image was

obtained from reference [1].

Figure 4. A modern CD spectropolarimeter by

JASCO Inc. This image was obtained from

reference [9].

Instrumentation of CD Spectroscopy

CD is measured by spectropolarimeters

The difference in the left and right

polarized light, circular dichroism, is measured

by instruments called spectropolarimeters

(figure4) [1]. These instruments convey the

measured circular dichroism in terms of

ellipticity (𝜃) in degrees as a function of

wavelength of light. Note, 𝜃 = tan-1(𝑏

𝑎) and b

and a represent the minor and major axis of the

ellipse produced[1]. The 𝜃 values may be

converted to ∆𝐴 using the formula[1]:

𝜃 = 32.98(∆𝐴)

To measure circular dichroism,

spectropolarimeters employ three different

approaches. The most common approach is

modulation, in which the incident radiation is

continously switched between the left and right

components by a modulator [1]. The alternating

radiation is detected by a photomultiplier[1].

The second approach is direct substraction. In

this approach the absorbances of both

components is measured separately and

subtracted from each other [1]. The last

approach is the ellipsometric approach, in

which the ellipticity of the transmitted radiation

is measured [1]. Detailed protocols for obtaining

CD measurements with spectropolarimeters are

provided in references [1], [15-17].

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Biological Applications

The most common application of CD is

the study of biomolecular structure. Due to the

introductory nature of this article only a brief

introduction to the study of proteins via CD

spectroscopy is provided. For other biological

applications consult references [1], [18] and

[19].

CD elucidates protein structure

Proteins are known to commonly have

chromophoric components in an asymmetric

environment and thus are ideal candidates for

CD spectroscopy [9]. They can be analysed over

a broad range of wavelengths, incorporating the

far UV region (<250 nm), near UV region

(>250 nm) and even the visible region [9]. The

far UV region (<250 nm) provides information

about the secondary structures of the proteins

such as 𝛼-helix, 𝛽-sheet or random coil [9].

These features are summarized in Figure 5.

The obtained spectra may be qualitatively

examined to determine contributions of each of

the structures for a given protein, or

deconstructed for a more quantitative picture [9].

In contrast, the near UV region (>250 nm)

offers more complex information regarding the

protein structure. This information is based on

chromophoric groups on phenylalanine,

tyrosine, tryptophan and cysteine [9]. The visible

region serves to provide information regarding

ligand-metal interactions and thus ideal for

examination of metalloproteins [9]. The CD

spectra for two proteins, calmodulin and EcoRI

endonuclease are shown in figure 6.

CD can detect structural changes

In addition to providing structural

information, the sensitivity of CD spectroscopy

can be utilized in the examination of changes in

structure. In particular, diverse conditions (pH,

temperature and etc.) can be employed to

monitor the impact on the CD spectrum [1] [9].

This application essentially translates to an easy

way to monitor structural stability of the

molecule of interest [9]. This type of application

was employed by Imperiali [3].

CD vs. X-Ray & NMR approaches

Circular Dichroism is a low resolution

technique providing information on the overall

structural features of the molecule being

inspected [1]. On the other hand, X-ray

crystallography and NMR are high resolution

techniques providing information at the atomic

level [1]. However, CD is a more efficient

technique requiring less time and a smaller

sample size. A high quality CD spectra of a

protein may be obtained on less than 0.1 mg in

the far UV range or 1 mg in the near UV and

visible range in 30 minutes or less [1]. In

contrast, x-ray crystallography and NMR are

relatively demanding techniques with specific

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requirements. X-ray crystallography requires

crystals of the protein being inspected while

NMR requires high concentrations of the

protein, usually about 0.5 mM, and is hence

limited to protein fragments or small proteins

[1]. CD also offers unmatched versatility,

allowing exploration of protein structure under

a range of experimental conditions and

measurements of rates at which structural

changes occur [1].

Figure 5. . The far UV region (<250 nm) provides

information about the secondary structures of the

proteins such as α-helix, β-sheet or random coil.

This image was adapted from reference [9].

Figure 6. The CD spectra in the far UV region for

calmodulin and EcoRI endonuclease. This image

was adapted from reference [20].

Conclusion

In conclusion, Circular Dichorism refers

to the differential absorption of left and right

circularly polarized light [5]. It is a versatile

technique that may be employed in the study of

proteins, nucleic acids and carbohydrates.

Although lacking in resolution relative to NMR

and X-ray approaches, this technique offers

powerful sensitivity and efficiency. It is capable

of producing high quality spectra for protein

samples as small as 0.1 mg (far UV) and 1 mg

(near UV/visible) in as little as 30 minutes [1].

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