The primary amino acid sequence of the protein contributes to the final conformational resolution of secondary and tertiary structures. Assessing biophysical properties, such as secondary and tertiary structures and thermal stability, provides information about these higher order structures and overall protein conformations.
At Creative Biolabs, protein-based drug conformation, along with the biophysical characterization of proteins, constitutes a high-order structure of biology, assessing the biophysical structure, function, and stability of biological macromolecules such as proteins, nucleic acids, lipids, and their complexes. Characterization techniques we offer include circular dichroism, differential scanning fluorescence, differential scanning calorimetry, dynamic light scattering and UV/visible spectroscopy. The kinetics and its iterative thermodynamic parameters (microscale thermophoresis, surface plasmon resonance, and isothermal titration calorimetry) are also characterized according to the specific needs of the customers.
| Services | Techniques |
| Secondary structure | Fourier transform infrared (FTIR) spectroscopy, circular dichroism |
| Tertiary structure | Near-UV circular dichroism (near-UV CD) spectroscopy, differential scanning calorimetry, X-ray crystallography |
| Thermal stability | Differential scanning calorimetry (DSC) |
Fourier Transform Infrared (FTIR) Spectroscopy
Secondary structure analysis examines hydrogen bond-mediated interactions between residues. The most common structural motifs of protein secondary structures are alpha-helices and beta-sheets. The secondary structure of the β-sheet-containing protein is elucidated mainly by Fourier transform infrared (FTIR) spectroscopy, through measuring the absorbing substance in the amide I region, whereas that of a protein containing predominantly alpha-helices is clarified typically by using far ultraviolet circular dichroism (far ultraviolet CD) spectroscopy.
Circular Dichroism
The tertiary structure of a protein depends on various interactions between different regions of the molecule, including hydrogen bonding, hydrophobic interactions, and salt bridges. Near-ultraviolet CD spectroscopy is commonly used to elucidate the tertiary structure of proteins, in which the average residue ellipticity is evaluated as a function of wavelength. The aromatic residue is absorbed between about 250-350 nm, primarily determined by the local environment of the aromatic residues (solvents exposed to or buried in the protein structure).
Differential Scanning Calorimetry (DSC)
The thermal stability of a protein (or a specific region of a protein) is regulated by the folding patterns of the protein; therefore, the evaluation of this feature reveals information about the conformation of the protein. Thermal stability is typically assessed using differential scanning calorimetry (DSC), which measures excess heat capacity as a function of temperature. As the temperature increases, the protein unfolds at the characteristic hot melting temperature, depending on the thermodynamic stability of the folded protein (or folded protein domains).
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