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Circular Dichroism Spectroscopy Analysis Service-Protein Structure Elucidation

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What Is Circular dichroism (CD) spectroscopy

Since the pioneering work of Kendrew, who utilized X-ray technology to unveil the folded structure of myoglobin, protein research has evolved from the determination of amino acid residue sequences to the in-depth exploration of spatial structures and conformations. It has become increasingly apparent that understanding the conformation of proteins and their related variations is of paramount significance for comprehending their physiological functions.

Circular dichroism (CD) spectroscopy has emerged as a rapid, straightforward, and reasonably accurate method for the investigation of protein conformations. In 1969, Greenfield was among the pioneers in utilizing CD spectroscopic data for estimating protein conformations, and subsequent research methodologies have been consistently documented. Significantly, over the last two decades, the analysis of protein secondary structures using far-ultraviolet CD (Far-UV CD) data has experienced notable advancements. These advancements encompass not only computational methods and fitting procedures but also coincide with the progress in X-ray crystallography and nuclear magnetic resonance techniques. Consequently, this progress has facilitated the precise determination of protein conformations, enhancing the accuracy of CD data fitting through more comprehensive databases.

Furthermore, researchers have unveiled distinctive advantages in the exploration of protein tertiary structures through CD spectroscopy. Specialized methods and accompanying software have been devised for the identification of protein tertiary structures employing far-ultraviolet CD spectroscopy. Additionally, near-ultraviolet circular dichroism (Near-UV CD), renowned for its sensitivity as a spectral probe, has the capacity to discern alterations in the microenvironment surrounding aromatic amino acid residues and disulfide bonds within proteins. Consequently, CD spectroscopy, serving as an indispensable instrument for the examination of protein or peptide conformation in solution, has engendered considerable interest among the scientific community.

Circular Dichroism Spectroscopy Analysis Service-Protein Structure Elucidation

Principles of Circular Dichroism

When a beam of plane-polarized light traverses a chiral medium, the refractive indices of the medium for left and right circularly polarized light differ. As a result, the velocities of these two components through the medium become distinct, leading to a change in the polarization direction of the superimposed plane-polarized light and the generation of optical rotation. Simultaneously, due to the differing molar absorptivities of the chiral medium for left and right circularly polarized light (εL(λ), εR(λ)), a disparity arises, Δε(λ) = εL(λ) - εR(λ). This leads to variations in the amplitudes of left and right circularly polarized light after passing through the medium, resulting in their superposition as elliptically polarized light. This phenomenon is referred to as circular dichroism.

Figure 1: Schematic Representation of Circular Dichroism PrinciplesFigure 1: Schematic Representation of Circular Dichroism Principles

Proteins are biomacromolecules with specific structures, comprised of amino acids linked by peptide bonds. They possess several structural levels, including primary, secondary, tertiary, and quaternary structures, and may also exhibit structural domains or supersecondary structures. In proteins and peptide molecules, peptide bonds, aromatic amino acid residues, and disulfide bridges within the peptide chain serve as the primary optically active chromophores. When plane-polarized light passes through, these chromophores exhibit differential absorption of left and right circularly polarized light, resulting in a discrepancy in the amplitude of the polarization vector. This transforms circularly polarized light into elliptically polarized light, giving rise to the phenomenon known as protein circular dichroism.

Primary Applications of Circular Dichroism Spectroscopy

Determination of Protein Secondary and Tertiary Spatial Structures

Proteins, as one of the three fundamental components of living organisms, play vital roles in various physiological processes. Circular dichroism (CD) spectroscopy scanning analysis holds significant importance in the study of protein secondary and higher-order structures. CD spectra in the far-ultraviolet region provide information about the arrangement of peptide bonds within proteins, allowing the calculation of the proportions of protein secondary structures such as α-helices, β-sheets, turns, and irregular coils. Moreover, CD spectra in the near-ultraviolet region reveal the distribution of amino acid residues with chromophoric side chains, including tryptophan, phenylalanine, tyrosine, and variations in the disulfide bond environment.

Investigation of Protein Thermal Denaturation and Thermodynamic Parameters

Under specific physical and chemical conditions, proteins can undergo conformational changes leading to inactivation. Therefore, the study of protein conformation and structural changes is of significant value. Thermal denaturation, often a common cause of protein unfolding, can be thoroughly investigated using a CD spectrophotometer equipped with a temperature control system. This allows the observation of the entire thermal denaturation process and the determination of the temperature (Tm value) at which protein denaturation occurs. Such studies are of paramount scientific importance for understanding the thermal stability of proteins and their denaturation processes when exposed to changing environmental conditions.

Our Circular Dichroism Spectroscopy Analysis Platform

Creative Proteomics has established a Circular Dichroism (CD) spectroscopy analysis platform based on the CD spectrophotometer for the investigation of the secondary structure and tertiary structure of proteins and peptide-based biological products. The instrument is equipped with temperature control and titration modules, allowing for in-depth research into the impact of factors such as temperature, pH, and ionic strength on protein folding states and stability.

Circular Dichroism Spectroscopy Analysis Workflow

Circular Dichroism Spectroscopy Analysis Workflow

Sample Requirements

Sample Types: Protein, Nucleic Acid Samples

Sample Forms: Liquid, Dry Powder

Storage Conditions: Low Temperature (Dry Ice)

Case Study 1: Characterization of Protein Higher-Order Structures The secondary structure of protein samples

Figure 2: Far-Ultraviolet Circular Dichroism Spectrum of ProteinFigure 2: Far-Ultraviolet Circular Dichroism Spectrum of Protein
Structure Proportions
Helix xx.9%
Antiparallel x.0%
Parallel x.5%
Beta-turn xx.2%
Random Coil xx.4%
Total Sum 100%

Table 1: CDNN Software for Protein Secondary Structure Prediction (190nm-260nm) Protein Sample Tertiary Structure

Figure 3: Near-Ultraviolet Circular Dichroism Spectrum of ProteinsFigure 3: Near-Ultraviolet Circular Dichroism Spectrum of Proteins

This spectral profile captures the absorption characteristics of aromatic amino acid residues, including tryptophan, phenylalanine, and tyrosine, and also reveals changes in the microenvironment of disulfide bonds. The influence of the surrounding environment imparts nuanced circular dichroism signals within this specific wavelength range. Monitoring alterations in these signals offers valuable insights into modifications in the protein's tertiary structure.

Case Study 2: Characterization of Nucleic Acid Higher-Order Structures

Figure 4: Circular Dichroism Spectra of mRNA at Different Temperature GradientsFigure 4: Circular Dichroism Spectra of mRNA at Different Temperature Gradients

With the elevation of temperature, the higher-order structure of mRNA undergoes disruption, resulting in a pronounced decline in the circular dichroism signal, accompanied by a concurrent shift in the peak position.

The Biological Analysis and Characterization team at Creative Proteomics brings forth extensive experience, with active involvement in the development and validation of diverse biological analysis methodologies. These encompass macromolecular protein drugs, monoclonal/polyclonal antibody drugs, antibody-drug conjugates (ADCs), nucleic acid drugs, glycosylation, charge heterogeneity, molecular size, and more. Within our specialized biological analysis laboratory, we are equipped with cutting-edge instrumentation, including LC-Q-TOF, LC-MS/MS, GC-MS, HPLC/UPLC, capillary electrophoresis, enzyme immunoassay, and circular dichroism spectrophotometers. Creative Proteomics offers a comprehensive, all-encompassing platform for research, analysis, and services related to biological products, adhering to international quality standards, and delivering tailored solutions for our clients' diverse needs in biological analysis and characterization.

Q: What information do circular dichroism spectra obtained in the near-ultraviolet and far-ultraviolet regions convey?

A: Circular dichroism spectra in the far-ultraviolet region offer insights into the secondary structure of the protein. These spectra illuminate the spatial orientation of peptide bonds within the protein, facilitating the calculation of the proportions of distinct secondary structural elements, such as α-helices, β-sheets, turns, and irregular coils. This data is pivotal for a comprehensive understanding of the protein's structural attributes and stability.

Conversely, circular dichroism spectra in the near-ultraviolet region provide details regarding the protein's side chains. They depict the spatial arrangement of amino acid residues containing chromophoric side groups, such as tryptophan, phenylalanine, and tyrosine, within the protein's structure. Furthermore, near-ultraviolet scans can unveil alterations in the microenvironment of disulfide bonds within the protein, a valuable resource for the investigation of the protein's functional attributes and interactions.

Q: What software is employed for the analysis of protein secondary structure, and what are its merits?

A: Currently, there exist multiple software alternatives for the analysis and prediction of protein secondary structure. Among these, CDNN stands out as a secondary structure prediction software rooted in deep neural network technology. Its capabilities encompass the prediction of a protein's secondary structure composition through the analysis of its amino acid sequence.

CDNN presents several advantages when compared to other software solutions:

High Precision: CDNN yields remarkably precise calculations, facilitating accurate predictions of a protein's secondary structure.

Expediency: CDNN boasts swift computation capabilities, enabling the expeditious processing of extensive protein sequence datasets.

Cost Efficiency: CDNN offers a cost-effective solution, resulting in savings in both analysis costs and time.

The software has garnered validation and application across various biomedical domains, including drug design and protein structure prediction.

Reference

  1. YAO H, WYNENDAELE E, XU X, et al.Circular dichroism in functional quality evaluation of medicines.J Pharm Biomed Anal, 2018

*For Research Use Only. Not for use in the treatment or diagnosis of disease.

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