Question

Compare and discuss the relative advantages and disadvantages to using circular dichroism or fluorescence spectroscopy or light scattering to study

1. protein folding

2. protein-ligand interactions

3. protein structure

Answer 1

Circular Dichroism ( CD)

• The secondary structure of protein can be determined using this method.
• Environment in which the protein present alters the conformation of the protein.
• Eg . Increase/decrease in temperature causes protein degradation .Similarly pH effects are also have profound effects on protein causing conformational change which makes changes in protein-ligand interaction .
• This change can be best viewd by circular Dichorism.
• Protein ligand interactions can also be viewed in real time by CD. For viewing this CD is used in stop and flow manner .
• Highly concentrated protein solutions with high salt , high buffer cannot be viewed in CD . This is because , the solution should be transparent to UV light. If there is high concentration of salt , UV cannot pass through and unable to detect the structure of protein.
• CD's application is limited because it cannot determine tertiary structure of protein.

Fluroscence spectroscopy

• In the fluroscence spectroscopy , the physical and chemical properties of the protein can be studied using this method.
• Tryptophan and tyrosine can exhibit furoscence naturally. Because of this property , the protein dynamics and protein ligand interactions can be seen.
• Fluroscent probe is attached specially and so that the particular reaction with protein ligand interactions can be studied.
• Fluroscent spectroscopy is highly sensitive , so that even if the protein is present in small quantities the dynamics and protein -ligand interaction can be studied.
• This spectroscopy enables the protein to detect its kinetics ,stability and thermodynamics of a protein.
• Even though it measures the protein structure , the environment in which it is present cannot be detected accurately and the results cannot be prperly interpreted.

Light - Scattering technique

• This technique is non -destructive, non -invasive and it is absolute compared to other techniques.
• The results acn be interpreted easily and it is more reliable.
• The size of the protein can be detected in real time.
• This detects very small proteins that cannot be detected by electron microscopy and other spectroscopic techniques.
• It is very useful in studying the protein dynamics , its interaction with movement and sub cellular location.
• Three dimensional structure of a particular protein can be studied .
• hence , this technique is widely used compared to others.