Circular Dichroism (CD) Overview
Circular Dichroism (CD) Overview
Further Reading
Several textbooks are recommended for comprehensive understanding:
Exploring Proteins. Price & Nairn (2009) - Oxford University Press
How Proteins Work. Williamson (2011) - Garland Science
Introduction to Protein Science. Lesk (2010, 2nd ed.) - Oxford University Press
Definition of Circular Dichroism
Circular Dichroism (CD): The differential absorbance of left (L) and right (R) circularly polarized light.
Sensitive to chirality and molecular asymmetry.
CD occurs in the same spectral regions as absorbance.
Understanding Circularly Polarized Light
Characteristics of Circularly Polarized Light
Circularly Polarized Light: A result of combining two plane-polarized light waves with a specific phase relationship.
Tutorial on Circular Polarization
Tutorial illustrating the oscillation of electric fields in plane-polarized light:
Horizontal Plane
Vertical Plane
Superposition of Plane-Polarized Light
When two waves of equal amplitude, wavelength, and phase in perpendicular planes interact:
Results in plane-polarized light at 45 degrees to the component waves.
Waves with a 90 degrees phase difference produce circularly polarized light.
A diagram may show the sum of two electric vectors (red and green) represented by a cyan line.
Phase Difference: Right and Left Circularly Polarized Light
Phase Difference +90°: Produces Right Circularly Polarized Light.
Phase Difference -90°: Produces Left Circularly Polarized Light.
Absorption Properties of Light
Absorption of Plane Polarized Light
When plane-polarized light passes through a sample:
The amplitude of the light leaving the sample is lower than that entering, indicating absorption.
Interaction of Light with Matter plays a vital role.
Absorption of Circularly Polarized Light
Similar principles apply; the amplitude of the circularly polarized light that exits is smaller than that which entered.
Circular Dichroism in a Medium
Materials that absorb L and R circularly polarized light differently exhibit Circular Dichroism (CD).
In the example provided, right-circularly polarized light (green) is absorbed more than left (red), resulting in elliptically polarized light.
The twist direction of light depends on the component absorbed the least (left-polarized light in this case).
CD Instrumentation
Components of a CD Instrument
Basic components include:
Sample
Detector: Measures light intensity.
Photoelastic modulator (PEM): Converts the linear polarized beam into LCP and RCP light oscillating at 50 Hz.
If the sample is CD active, the relative absorbance of LCP and RCP will differ, producing a measurable signal oscillation.
Monochromator and Linear Polarizer: Allows light selection at various wavelengths, aiding in obtaining the CD spectrum.
CD Spectrum Generation
The signal changes with wavelength enables a full CD spectrum to be recorded, displaying light intensity vs. time.
Measurement Units for Circular Dichroism
CD Measurement Definition:
ext{CD} = ext{differential absorbance} = ext{ΔA} = A{LCP} - A{RCP}
Parameters needed:
Path-length of the cuvette (l, in cm)
Protein concentration (c, in M)
Molar Circular Dichroism Equation:
ext{Δε} = ε{LCP} - ε{RCP}
Δε indicates the molar extinction coefficient difference and has units of M$^{-1}$ cm$^{-1}$.
Common Reporting Units
Some spectrometers report ΔA using angles:
ext{ΔA} = heta / 32982 (when θ is measured in millidegrees).
Mean Residue Molar Circular Dichroism:
ext{Δε}_{MR} = rac{Δε}{N} = rac{ΔA}{(c imes l imes N)}
Where N denotes the number of residues, facilitating comparison of CD spectra across proteins of different molecular weights.
CD Spectra in Proteins
Far-UV CD Spectra (190-250 nm)
Provides information primarily on peptide bonds.
Characteristic spectra depend on the structural organization (α-helix, β-sheet, or random coil).
Sensitivity is best for α-helix structures.
Protein concentrations typically required: 0.2-1.0 mg/ml (0.1-0.5 mg of protein in a 0.5 ml volume cuvette).
Near-UV CD Spectra (250-300 nm)
Focuses on aromatic side chains.
Aromatic side chains in a folded protein have an asymmetric environment; less mobility results in a stronger CD signal.
No strict rules relate environment to spectrum, serving as a useful fingerprint for tertiary structure.
Requires higher protein concentrations (0.5 - 2 mg/ml) as absorption is weaker.
Specific absorbance order: Trp > Tyr > Phe.
Biological Applications of CD
Applications Overview
Estimating Secondary Structure Content.
Measuring Protein Unfolding and Refolding.
Determining Structural Authenticity.
1. Estimating Secondary Structure Content
Initially approached through multi-component analysis of reference spectra for three types of secondary structures.
More recent methodologies utilize reference datasets containing CD spectra from proteins of known 3D structures, allowing comparison with proteins of unknown structure to find matches.
Expansion of reference databases includes theoretical CD spectra predictions from high-resolution 3D structures, providing broader coverage of protein folds.
Tools available online include K2D2 and K2D3.
2. Measuring Protein Unfolding and Refolding
CD measurements taken at 222 nm show negative peaks indicating α-helix presence, monitoring the unfolding (solid symbols) and refolding (open symbols) of specific proteins under various conditions.
Panel A: Shows heat denaturation; Panel B: Shows chemical denaturation using guanidinium chloride and urea, allowing the determination of protein conformational stability.
Indications of buffer effects on protein stability, showcasing condition A advantageously over condition C.
3. Determining Structural Authenticity
Near-UV CD demonstrates subtle differences in protein conformations compared to far-UV CD, revealing reproducible changes in monoclonal antibodies at 240 nm, correlating with stability.
Far-UV CD used to compare proteins from natural sources vs. recombinant sources (e.g., E. coli), highlighting significant spectral differences, particularly at 220 nm, indicating potential misfolding in recombinant proteins.