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What is Circular Dichroism?
Circular Dichroism (CD) is a sophisticated analytical technique used to study chiral molecules by examining their interaction with circularly polarized light. The essence of CD lies in the differential absorption of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL) by these chiral substances.
Chiral molecules are those that have non-superimposable mirror images, much like left and right hands. These molecules interact differently with circularly polarized light due to their asymmetrical structure. When chiral molecules absorb circularly polarized light, they do so with varying efficiencies depending on the direction of the polarization. This differential absorption can be expressed as:
Circular Dichroism = ΔA(λ) = A(λ)L-CPL ‐ A(λ)R-CPL, where λ is the wavelength
Mechanism of Circular Dichroism
The CD effect arises from the interaction of chiral molecules with circularly polarized light, which can be thought of as light that rotates in a helical pattern as it propagates. Chiral molecules can absorb this light in a manner that is dependent on the handedness of the light. This means that the interaction is not merely about the amount of light absorbed but also about how the molecular structure affects the light's polarization.
When a sample containing chiral molecules is exposed to circularly polarized light, the light's intensity changes differently depending on its polarization direction. By measuring these differences in absorption, researchers can infer various structural details about the molecules.
Types of Circular Dichroism Spectroscopy
UV Circular Dichroism
UV Circular Dichroism is employed primarily to analyze the secondary structure of proteins. By examining the far-UV region of the spectrum (typically 190-250 nm), this technique provides detailed information about the content and arrangement of secondary structural elements such as α-helices, β-sheets, and random coils. It is crucial for understanding protein folding, stability, and conformational changes.
UV/Vis Circular Dichroism
UV/Vis Circular Dichroism extends the analysis to the near-UV and visible regions of the spectrum. This type of CD spectroscopy is used to study charge-transfer transitions and electronic properties of molecules. It helps in probing molecular interactions, electronic transitions, and conformational changes, offering insights into the behavior of chromophores and other complex molecular systems.
Near-Infrared Circular Dichroism
Near-Infrared Circular Dichroism is used to explore the geometric and electronic structures of metal complexes. By focusing on the near-infrared region, this technique provides valuable information on metal d→d transitions and coordination environments, making it essential for studying transition metal complexes and other chiral metal-containing compounds.
Vibrational Circular Dichroism
Vibrational Circular Dichroism utilizes infrared light to examine the vibrational transitions of chiral molecules. This type of CD spectroscopy is particularly useful for structural studies of small organic molecules, proteins, and nucleic acids. It provides insights into the conformational characteristics and molecular vibrations, contributing to a deeper understanding of molecular structure and interactions.
Circular Dichroism Solutions in Creative Proteomics
Protein Secondary Structure Analysis
Understanding the secondary structure of proteins is essential for elucidating their function and stability. Our UV Circular Dichroism services are specifically designed to:
- Determine Protein Folding: Analyze the far-UV spectra (190-250 nm) to identify and quantify secondary structural elements such as α-helices, β-sheets, and random coils. This information is crucial for understanding protein folding and stability.
- Assess Protein Stability: Monitor changes in the secondary structure in response to environmental factors such as temperature, pH, or denaturants, providing insights into protein stability and conformational dynamics.
- Characterize Protein Conformational Changes: Evaluate how proteins undergo conformational changes upon ligand binding or other interactions, which is valuable for drug development and functional studies.
Nucleic Acid Structural Studies
- Investigate DNA and RNA Conformations: Examine the structural transitions between different forms of DNA (e.g., B-DNA to A-DNA) and RNA, providing insights into their functional states and interactions.
- Study Nucleic Acid-Ligand Interactions: Analyze how nucleic acids interact with ligands or other biomolecules, offering a detailed view of binding events and their effects on nucleic acid structure.
Charge-Transfer Transition Analysis
- Characterization of Electronic Transitions: Study charge-transfer transitions and electronic properties in various molecules. This is particularly useful for understanding complex molecular systems and their behavior under different conditions.
- Analysis of Molecular Interactions: Investigate how molecules interact with each other or with external factors, providing insights into conformational changes and electronic transitions.
Metal Complex and Coordination Studies
- Exploring Metal d→d Transitions: Study the geometric and electronic structures of metal complexes by analyzing the near-infrared region. This is essential for understanding the coordination environment and electronic configuration of transition metal complexes.
- Characterizing Chiral Metal-Containing Compounds: Investigate chiral metal complexes and their behavior, which is valuable for applications in catalysis, material science, and structural chemistry.
Vibrational Analysis
- Structural Characterization of Small Molecules: Examine vibrational transitions to gain insights into the structural details of small organic molecules, including their conformations and functional groups.
- Protein and Nucleic Acid Studies: Extend structural analysis to proteins and DNA, providing detailed information on their vibrational modes and conformational states.
Custom and Specialized Analyses
- Tailored Experimental Design: Develop custom CD spectroscopy experiments based on specific research needs, including special conditions or unique sample types.
Sample Requirements for Circular Dichroism Analysis
| Sample Type | Concentration | Volume | Purity | Solvent Compatibility | Preparation & Handling |
|---|---|---|---|---|---|
| Proteins | 0.1 to 1 mg/mL | 0.5 to 1 mL | Purified, free of aggregates | Compatible buffer solutions; avoid strong absorbers | Ensure protein is well-dissolved and homogeneous; avoid high concentrations of detergents unless required |
| Nucleic Acids | 5 to 50 µM | 0.5 to 1 mL | High purity, free from contaminants | Aqueous buffers like Tris-HCl or PBS; avoid strong absorbers | Prepare in a clean environment; remove residual solvents or contaminants if necessary |
| Small Molecules | 0.1 to 1 mM | 0.5 to 1 mL | Pure, free from impurities | Solvents with minimal absorption in the CD range | Dissolve or prepare to ensure a clear, stable solution; avoid particulates |
| Metal Complexes | 0.1 to 1 mM | 0.5 to 1 mL | Clean, stable form | Solvents that do not interact with the sample | Minimize oxidation or decomposition; prepare stable solutions |
Molecular dynamics ensemble refinement of intrinsically disordered peptides according to deconvoluted spectra from circular dichroism
Journal: Biophysical journal
Published: 2020
Background
Intrinsically disordered proteins/peptides (IDPs) lack stable structures and exist as ensembles of weakly ordered forms. They are crucial in biological processes but are implicated in diseases when misregulated. IDPs typically form stable structures only upon binding to targets, allowing them to interact with multiple proteins.
Traditional structural methods like cryo-EM and crystallography are unsuitable for IDPs. Solution-based techniques such as NMR provide averaged structures, and computational models often rely on parameters from stable proteins, which can be biased. Circular dichroism (CD) spectroscopy offers practical benefits but lacks high resolution. Combining CD with non-negative least-squares (NN-LSQ) fitting and all-atomistic MD simulations has improved the understanding of IDP structures and their functional implications.
Materials & Methods
Peptide Synthesis and Preparation: Three 20-amino-acid peptides based on the CaM-binding domain of CaMKII (residues 293–312) were synthesized by LifeTein LLC. The peptides had over 95% purity and were verified by mass spectrometry. Binding kinetics to calcium/CaM were previously measured using stopped-flow fluorimetry, revealing a 3000-fold decrease in affinity due to mutations (R296A/R297A/K298A).
CD Spectroscopy: Far-ultraviolet CD spectra were collected with a JASCO-815 spectrophotometer using 1.0 mm Suprasil cuvettes. Peptide solutions (100 μM in 10 mM Tris buffer, pH 7.5) were scanned between 190 and 260 nm at 20°C. Measurements were repeated three times with freshly prepared samples.
CD Data Analysis: CD spectra were deconvoluted using CDPro software with the SDP48 dataset and methods (CDSSTR, CONTIN/LL, SELCON3). Results were categorized into six secondary structure types, which were further consolidated into four main categories (helix, strand, turn, unordered) for comparison.
NN-LSQ Fitting: CD spectra were deconvoluted using NN-LSQ fitting with the SDP48 dataset. The fitting aimed to minimize the squared difference between experimental and reference spectra, using a non-negative linear combination of basis spectra. The secondary structure fractions were calculated based on the weight coefficients obtained from the fitting.
Validation of CD Deconvolution Deconvolution methods (NN-LSQ, CONTIN/LL, SELCON3, CDSSTR) were validated by comparing results with known secondary structures from the Protein Circular Dichroism Data Bank (PCDDB) using root mean square deviation (δ) and correlation (r) coefficients.
MD Simulations: Initial peptide structures were built using AMBERTOOLS 14 based on amino acid sequences, without N- and C-terminal capping. MD simulations were performed with AMBER 14, using the ff99sb force field and implicit solvent model. Simulations included energy minimization, temperature equilibration, and production runs at 277, 285, and 293 K for 80 ns, with a total of 2.4 μs simulation time per peptide.
Analysis of MD Trajectories: Secondary structure content was analyzed using CPPTRAJ in AMBERTOOLS, categorized into four main types. Frames from MD trajectories were selected based on similarity to CD deconvolution results.
Contact Map Analysis: Contact maps were generated for CD-guided MD structures, defining contacts based on distance and hydrogen bonding criteria between residues at least four residues apart.
Results
CD Spectra Analysis:
The secondary structure of AAA peptide shows significant differences from RRK and RAK, indicating a shift from disordered to more ordered structures. AAA peptide exhibits a mixed secondary structure rather than pure α-helical content.
Far-ultraviolet CD spectra of the CaMKII peptides.
Deconvolution Analysis:
Standard CD deconvolution methods (CDPro, CAPITO, BeStSel) produced inconsistent results with large RMSDs. NN-LSQ fitting, using denatured protein data, revealed an increase in β-sheet content for AAA compared to RRK and RAK.
Comparison between the fitting of the CD spectra using the CDPro and NN-LSQ fitting.
MD Simulations:
Simulations showed a bias towards helical structures and failed to match the secondary structure shifts observed in CD data. β-sheet structures were underrepresented in the simulations.
Contact probability map of the CD-refined MD structures.
Contact Map and Hydrogen Bond Analysis:
AAA peptide shows strong β-sheet formation with stable interactions between residues. Unique hydrogen bonding patterns in AAA support β-sheet structure formation, while RRK and RAK display different bonding patterns with less stability.
Reference
- Ezerski, Jacob C., et al. "Molecular dynamics ensemble refinement of intrinsically disordered peptides according to deconvoluted spectra from circular dichroism." Biophysical journal 118.7 (2020): 1665-1678.
What types of samples can be analyzed using CD?
CD spectroscopy can be applied to a variety of samples, including proteins, nucleic acids (such as DNA and RNA), polysaccharides, and synthetic polymers. The sample should be chiral and able to interact with circularly polarized light.
What information can be obtained from a CD spectrum?
- Secondary structure: Identification and quantification of structural elements like α-helices, β-sheets, and random coils.
- Conformational changes: Structural changes in proteins or nucleic acids due to mutations, binding events, or environmental changes.
- Molecular interactions: Binding interactions and conformational changes upon ligand binding.
How do you prepare a sample for CD spectroscopy?
For CD spectroscopy, samples are usually prepared in a suitable buffer, and the concentration should be optimized for the best signal-to-noise ratio. Proteins are typically dissolved at concentrations ranging from 1 to 100 µM, while nucleic acids are often at concentrations of 1 to 10 µM. The sample is placed in a quartz cuvette with an appropriate path length, usually between 0.1 and 1 cm.
What are the common types of CD spectra and their uses?
Far-UV CD (190-250 nm): Used primarily to analyze protein secondary structure (e.g., α-helices and β-sheets).
Near-UV CD (250-320 nm): Provides information about the tertiary structure and local environments of aromatic residues.
Optical Rotatory Dispersion (ORD): Sometimes used in conjunction with CD to study chiral properties of the sample.
What are some limitations of CD spectroscopy?
Sensitivity: CD spectroscopy might not detect very subtle structural changes or low-concentration samples.
Complexity: Interpreting CD spectra can be complex, especially when dealing with mixtures or overlapping signals from different secondary structures.
Dependence on Reference Data: Accurate analysis often requires reference spectra or deconvolution methods to interpret secondary structure content.
How is CD data analyzed?
CD data is typically analyzed by comparing experimental spectra to reference spectra or using computational deconvolution methods to estimate secondary structure content. Software tools and algorithms, like CDPro and BeStSel, can assist in interpreting the spectra and quantifying structural components.
Can CD spectroscopy be used to study dynamic changes in proteins?
Yes, CD spectroscopy can be used to monitor conformational changes over time or under different conditions (e.g., pH, temperature, or ligand binding). Real-time monitoring can provide insights into dynamic processes and structural transitions.
How does temperature affect CD spectra?
Temperature can influence protein and nucleic acid structures, potentially leading to conformational changes. CD spectra recorded at different temperatures can reveal information about thermal stability, unfolding, and structural transitions.
Is CD spectroscopy compatible with other techniques?
Yes, CD spectroscopy is often used in conjunction with other techniques, such as X-ray crystallography, NMR spectroscopy, and mass spectrometry, to provide a more comprehensive understanding of molecular structures and dynamics.
How do you interpret a CD spectrum?
- Identify the wavelength regions: Key features such as negative and positive bands can indicate specific secondary structures.
- Compare with reference spectra: Use spectra of known secondary structures as a reference.
- Use deconvolution software: Employ algorithms and tools (e.g., CDPro, BeStSel) to quantify secondary structure content based on the spectrum.
What factors can affect CD measurements?
- Sample concentration: Too high or too low concentration can lead to inaccurate readings.
- Path length: The thickness of the cuvette affects the intensity of the CD signal.
- Buffer conditions: pH, ionic strength, and buffer composition can influence the CD spectra.
- Temperature: Changes in temperature can alter protein conformation and CD signal.
How can you ensure accurate CD measurements?
- Calibrate the instrument: Regular calibration of the CD spectrometer is essential.
- Use appropriate controls: Include buffer and solvent controls to account for background signals.
- Standardize sample preparation: Consistently prepare samples and maintain proper concentration and buffer conditions.
Chemoproteomic identification of CO2-dependent lysine carboxylation in proteins.
King, D. T., Zhu, S., Hardie, D. B., Serrano-Negrón, J. E., Madden, Z., Kolappan, S., & Vocadlo, D. J.
Journal: Nature Chemical Biology
Year: 2022
https://doi.org/10.1038/s41589-022-01043-1
