9.2 Circular dichroism and optical rotatory dispersion

Circular dichroism and optical rotatory dispersion are powerful techniques for studying biomolecular structure. They measure how chiral molecules interact with polarized light, revealing crucial info about protein and nucleic acid conformations.

These methods are super useful for tracking changes in biomolecule shape and folding. They're non-destructive, need tiny samples, and can monitor shifts due to temperature, pH, or binding. CD and ORD are key players in understanding biological systems.

Circular Dichroism and Optical Rotatory Dispersion

Defining CD and ORD

Top images from around the web for Defining CD and ORD

• 

  Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original

  Is this image relevant?

• 

  Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original

  Is this image relevant?

• 

  Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original

  Is this image relevant?

• 

  Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original

  Is this image relevant?

1 of 2

Top images from around the web for Defining CD and ORD

• 

  Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original

  Is this image relevant?

• 

  Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original

  Is this image relevant?

• 

  Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original

  Is this image relevant?

• 

  Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original

  Is this image relevant?

1 of 2

• Circular dichroism (CD) measures the differential absorption of left and right circularly polarized light by chiral molecules providing information about the asymmetry of the molecule
• Optical rotatory dispersion (ORD) measures the wavelength-dependent rotation of the plane of linearly polarized light by chiral molecules offering insights into the molecule's chirality and structure
• CD and ORD are sensitive to the conformation of biomolecules, particularly proteins (secondary structure) and nucleic acids (base stacking) making them valuable tools for studying secondary structure, folding, and interactions
• These techniques are non-destructive, require small sample quantities (micromolar range), and can monitor conformational changes in response to environmental factors such as temperature, pH, and ligand binding

Relevance in Biophysical Studies

• CD and ORD provide valuable information about the secondary and tertiary structure of proteins and nucleic acids
• They can be used to study protein folding/unfolding, ligand binding, and oligomerization by tracking shifts in the intensity and position of characteristic spectral bands
• These techniques are often used in conjunction with other biophysical methods (X-ray crystallography, NMR spectroscopy) to provide a comprehensive understanding of biomolecular structure and dynamics
• CD and ORD are widely applied in the pharmaceutical industry for drug discovery and development to assess the conformational stability and binding interactions of drug candidates with their targets

Principles of CD and ORD Measurements

Interaction of Polarized Light with Chiral Molecules

• CD and ORD measurements rely on the interaction between polarized light and chiral molecules, which have non-superimposable mirror images called enantiomers
• Chiral molecules interact differently with left and right circularly polarized light due to their asymmetric structure
• The differential interaction of chiral molecules with polarized light forms the basis for CD and ORD measurements
• Examples of chiral biomolecules include amino acids (except glycine), sugars (glucose), and nucleotides (DNA and RNA)

Measuring CD and ORD Signals

• In CD, the difference in absorption of left and right circularly polarized light by a chiral molecule is measured as a function of wavelength resulting in a CD spectrum
• The CD signal arises from the differential absorption of the electric and magnetic components of the polarized light by the molecule's electronic transitions which are sensitive to the molecule's asymmetry and conformation
• In ORD, the rotation of the plane of linearly polarized light is measured as a function of wavelength providing information about the molecule's chirality and structure
• The ORD signal originates from the difference in refractive indices for left and right circularly polarized light which depends on the molecule's electronic transitions and conformation

Sensitivity to Biomolecular Structure

• The sensitivity of CD and ORD to biomolecular structure stems from the unique electronic transitions associated with the peptide backbone in proteins and the nucleotide bases in nucleic acids which are influenced by the molecule's secondary structure and higher-order conformations
• In proteins, the peptide bond absorbs in the far-UV region (190-250 nm) giving rise to characteristic CD signals for different secondary structure elements (α-helices, β-sheets, random coils)
• In nucleic acids, the base stacking interactions and the asymmetric sugar-phosphate backbone contribute to the CD signal in the near-UV region (250-300 nm) reflecting the conformation and tertiary structure

Interpreting CD and ORD Spectra

Secondary Structure Analysis of Proteins

• CD spectra of proteins in the far-UV region (190-250 nm) provide information about the secondary structure content as different secondary structure elements exhibit characteristic CD signatures
  • α-helices show a strong positive band at ~190 nm and negative bands at ~208 nm and ~222 nm
  • β-sheets display a negative band at ~215 nm and a positive band at ~198 nm
  • Random coils have a strong negative band at ~200 nm and a weak positive band at ~220 nm
• The relative intensities of these bands can estimate the fraction of each secondary structure element in a protein using deconvolution algorithms and reference datasets
• Changes in CD spectra can monitor conformational transitions, such as protein folding/unfolding, ligand binding, and oligomerization by tracking shifts in the intensity and position of the characteristic bands

Tertiary Structure and Conformational Changes

• Near-UV CD spectra (250-320 nm) reflect the tertiary structure of proteins arising from the asymmetric environment of aromatic amino acids (tryptophan, tyrosine, phenylalanine) and disulfide bonds
• Changes in near-UV CD spectra can indicate alterations in the tertiary structure of proteins due to ligand binding, mutations, or environmental factors (pH, temperature)
• ORD spectra provide complementary information to CD with the specific rotation at a given wavelength reflecting the overall chirality and conformation of the molecule
• Combined analysis of CD and ORD data can enhance the accuracy and reliability of structural insights derived from these techniques by cross-validating the results and identifying potential artifacts

Strengths and Weaknesses of CD vs ORD

Strengths of CD and ORD Techniques

• High sensitivity to changes in secondary and tertiary structure of proteins and nucleic acids enabling the detection of subtle conformational changes
• Non-destructive nature of the measurements allowing for the recovery of samples for further analysis or downstream applications
• Small sample quantities required (typically in the micromolar range) making them suitable for studying precious or limited samples (recombinant proteins, purified nucleic acids)
• Rapid data acquisition enabling real-time monitoring of conformational changes and kinetic studies of biomolecular processes (folding, binding)
• Applicable to a wide range of solution conditions (pH, temperature, ionic strength) facilitating the study of biomolecules under physiologically relevant conditions

Weaknesses and Limitations

• Low resolution structural information compared to techniques like X-ray crystallography and NMR spectroscopy limiting the atomic-level details that can be obtained
• Difficulty in interpreting spectra for complex systems with multiple conformational states or intermolecular interactions requiring additional experiments or computational modeling
• Potential artifacts arising from sample preparation (aggregation, impurities), buffer composition (absorbing compounds, high salt), and instrument calibration (baseline drift, polarization artifacts) necessitating careful experimental design and data validation
• Limited applicability to non-chiral molecules or those with weak electronic transitions resulting in low signal-to-noise ratios or featureless spectra
• Requirement for specialized instrumentation (CD spectrometers) and expertise in data analysis and interpretation which may not be readily available in all research settings Despite these limitations, CD and ORD remain valuable tools for studying biomolecular structure and dynamics particularly when used in conjunction with other biophysical techniques (fluorescence spectroscopy, calorimetry) to provide a comprehensive understanding of the system.