9.2 Circular dichroism and optical rotatory dispersion
5 min read•august 1, 2024
Circular dichroism and are powerful techniques for studying biomolecular structure. They measure how 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
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Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original
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Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original
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Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original
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Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original
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Top images from around the web for Defining CD and ORD
Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original
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Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original
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Fluorescence detected circular dichroism (FDCD) for supramolecular host–guest complexes ... View original
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Reversal aggregation-induced circular dichroism from axial chirality transfer via self-assembled ... View original
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- 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 and structure
- CD and ORD are sensitive to the conformation of , 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 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.
Key Terms to Review (16)
Biomolecules: Biomolecules are large, complex molecules that are essential for life, including proteins, nucleic acids, carbohydrates, and lipids. These molecules play crucial roles in biological processes and can exhibit unique optical properties when interacting with light, which is key to understanding their structure and function.
Brewster Angle: The Brewster angle is the angle of incidence at which light with a particular polarization is perfectly transmitted through a transparent dielectric surface, with no reflection. At this angle, the reflected light is completely polarized perpendicular to the plane of incidence. Understanding the Brewster angle is essential in studying how light interacts with materials, particularly in techniques like circular dichroism and optical rotatory dispersion.
Chiral molecules: Chiral molecules are molecules that cannot be superimposed on their mirror images, meaning they exist in two distinct forms, known as enantiomers. This property of chirality is important because the two enantiomers of a chiral molecule can have vastly different biological activities and physical properties. The behavior of chiral molecules in light interaction leads to unique phenomena like circular dichroism and optical rotatory dispersion.
Chirality: Chirality is a geometric property of molecules that refers to their non-superimposable mirror images, much like left and right hands. This feature is crucial in chemistry, particularly in the behavior of chiral molecules in biological systems and their interactions with light. Chirality plays a key role in the study of optical activity, where chiral substances can rotate plane-polarized light, and is fundamental to understanding phenomena such as circular dichroism and optical rotatory dispersion.
Circular dichroism spectrometer: A circular dichroism spectrometer is an instrument used to measure the differential absorption of left-handed and right-handed circularly polarized light by optically active substances. This technique is particularly useful for studying the secondary structure of proteins and other chiral molecules, providing insights into their conformational changes and interactions. By analyzing how these substances absorb light at different wavelengths, researchers can glean important information about molecular structure and dynamics.
Circular Dichroism Spectroscopy: Circular dichroism spectroscopy is a technique used to measure the differential absorption of left-handed and right-handed circularly polarized light by optically active substances, providing insight into their structural characteristics. This method is particularly valuable in studying biomolecules, as it can reveal information about their secondary structures, such as alpha-helices and beta-sheets, through their unique optical activity. It connects closely with principles of molecular chirality and interactions of light with matter, making it a key tool for understanding the conformational properties of complex biological molecules.
Cotton Effect: The Cotton Effect refers to the phenomenon observed in circular dichroism (CD) spectroscopy where the optical rotation of chiral molecules differs when measuring their absorption of left-handed and right-handed circularly polarized light. This effect is particularly significant in the study of chiral compounds, such as proteins and nucleic acids, as it provides insights into their conformational states and interactions. The Cotton Effect arises from the differential absorption of light due to the structural asymmetry of the molecules, making it a crucial aspect in understanding molecular chirality.
Electromagnetic radiation: Electromagnetic radiation is a form of energy that travels through space at the speed of light and encompasses a wide range of wavelengths and frequencies, including visible light, ultraviolet light, infrared radiation, radio waves, and X-rays. This type of radiation plays a crucial role in various spectroscopic techniques, allowing researchers to gain insights into molecular structures and interactions based on how molecules absorb and emit light.
J. A. Pople: J. A. Pople, or John Andrew Pople, was a prominent British chemist known for his groundbreaking contributions to computational chemistry, particularly in developing methods for quantum chemistry calculations. His work revolutionized the field by making complex quantum mechanical calculations more accessible to chemists and contributed significantly to understanding molecular structures and behaviors through computational techniques.
Molar ellipticity: Molar ellipticity is a measure of the degree of optical activity of chiral molecules in solution, defined as the specific rotation of a substance multiplied by its molar concentration. It provides insights into the structure and conformation of biomolecules, particularly proteins and nucleic acids, by analyzing how they interact with polarized light. Understanding molar ellipticity is essential in techniques like circular dichroism and optical rotatory dispersion, where it helps to elucidate molecular characteristics and behaviors.
Optical activity detector: An optical activity detector is an instrument used to measure the rotation of plane-polarized light as it passes through a chiral substance. This rotation is a result of the interactions between light and chiral molecules, which can provide valuable insights into their structural properties and concentration. By analyzing the degree of rotation, researchers can deduce information about the molecular configuration and stereochemistry of the sample being studied.
Optical rotatory dispersion: Optical rotatory dispersion refers to the phenomenon where the rotation of the plane of polarized light occurs when it passes through a chiral substance, and this rotation varies with the wavelength of the light used. This property is closely related to circular dichroism, where chiral molecules interact differently with left-handed and right-handed circularly polarized light. Understanding optical rotatory dispersion is essential for studying the structural characteristics of biomolecules and other chiral compounds.
Protein secondary structure analysis: Protein secondary structure analysis refers to the assessment and characterization of the local spatial arrangement of the polypeptide backbone in proteins, specifically focusing on elements like alpha-helices and beta-sheets. This type of analysis helps in understanding the stability, folding, and function of proteins, providing insights into how they interact with other molecules. Techniques such as circular dichroism and optical rotatory dispersion play a significant role in this analysis, as they help identify the presence and content of these structural features.
Specific rotation: Specific rotation is a measure of how much a substance rotates the plane of polarized light, defined as the observed rotation of polarized light divided by the concentration of the optically active substance and the path length through which the light passes. This property is essential for understanding molecular chirality and plays a significant role in techniques that analyze the optical properties of chiral molecules, like circular dichroism and optical rotatory dispersion.
Studying nucleic acid conformation: Studying nucleic acid conformation involves analyzing the three-dimensional structure and arrangement of nucleic acids, such as DNA and RNA, which can influence their biological functions. Understanding these conformational states is critical because they affect processes like replication, transcription, and translation. Different methods, including spectroscopy techniques, are used to investigate these conformations, providing insights into the stability and interactions of nucleic acids within biological systems.
William E. G. Müller: William E. G. Müller is a notable figure in the field of biophysical chemistry, particularly recognized for his contributions to the understanding of circular dichroism and optical rotatory dispersion. His work has significantly advanced the methodologies and applications of these techniques, enabling deeper insights into molecular structures and interactions, especially in biological systems. His research has paved the way for many important developments in spectroscopy.