What is the secondary structure?
What is the secondary structure? Are there any differences in overall 2o or 3o structure with another similar protein or between the wild type and a recombinant protein?
Circular Dichroism (CD) measures the differences in absorption of circular polarised light. It is routinely used to measure the secondary and tertiary structure of proteins and nucleic acids. Analysis of proteins in the far UV region between 185-260nm allows differential absorption to be measured due to the chirality of the peptide bond alignment. Data obtained can be helical, beta sheet and random coil content of the protein. Analysis in the near UV region, between 250-320nm allows the measurement of contributions from aromatic amino acids residues provided they are held rigidly in an asymmetric environment. In other words if the aromatic residues are placed in a disordered region of the protein or the protein is in a molten globular state these contributions will not be measurable by CD. Other protein chromophores and disulphide bonds can also contribute to protein CD spectra by virtue of their own chirality or induced chirality when bound to or present in the protein. Similarly small molecule binding to proteins and nucleic acids can be measured using far UV 195-250nm.
Fourier Transform InfraRed Spectroscopy can be extremely useful for protein and DNA/RNA characterisation, ligand interactions and for analysing protein dynamics particularly in membrane environments. The technique generally involves monitoring spectral contributions from intrinsic probes e.g. the amide I bond C=O stretching associated with the protein backbone, the C=O stretch of carboxylic acids involved in proton transfer reactions as well as vibrations associated with solvents and ligands. Different lipid compositions give rise to distinct spectral profiles in the C−H stretching region. The technique is particularly useful for monitoring changes in protein secondary structure since different secondary structural contributions occur in distinct regions of the spectrum (by contrast in the case of Circular Dichroism spectroscopy the beta-sheet contributions are often swamped by the larger intensities of alpha-helical contributions).
CD Sample Criteria
The material should be chiral, homogenous and the buffer or solvent should not absorb below 200nm. High salt concentrations and chloride ions should be avoided if possible
For far UV CD 185-250nm Typical sample 300ul of 0.1-0.8mg/ml.
For near UV CD 250-320nm Typical sample required 1ml of 1mg/ml.