Da Yu Protein Sciences – Biophysical Characterization
Circular Dichroism Spectroscopy - Circular dichroism (CD) spectroscopy measures the difference in the absorption of left and right circularly polarized light:
Δ A(λ) = A(λ)L – A(λ)R
The difference in absorbance is due to molecular asymmetry, a characteristic of chiral molecules. Chiral molecules are enantiomers, which are isomers that are not superimposable on their mirror images. All of the 20 common amino acids, except glycine, are chiral molecules. In proteins, the amide bond connecting neighboring amino acids is a chromophore in the far UV region (< 250 nM). These chromophores exist in structured arrays in the polypeptide backbone that have differential interactions with light. These molecular properties make CD spectroscopy a powerful and versatile tool for the biophysical characterization of proteins and it has the following capabilities:
- Estimation of protein conformation – The relationship of neighboring amide bonds in the polypeptide backbone can lead to estimations of protein secondary structure for α-helixes, β-sheets and random coils in the far UV region using appropriate algorithms.
- Measurement of protein conformational changes - Changes in protein secondary or tertiary structure induced by environmental perturbations (e.g., pH, temperature) can provide insight into protein stability.
- Determination of binding constants – When a ligand binds to a protein, the protein’s stability is increased. This increased stability can be measured by inducing protein unfolding through environmental changes (e.g., temperature, chemical denaturation) that perturb the CD signal quantitatively to the extent that ligand-binding constants can be determined.
- Calculation of protein unfolding thermodynamics – CD can monitor protein unfolding and produce an unfolding constant, K = [Unfolded]/[Folded], that can be used to determine the free energy (ΔG) of unfolding (ΔG = nRTlnK).
Da Yu Protein Sciences offers all these CD spectroscopy services to our clients. For more information on these or our other biophysical laboratory services please Contact Us.