Circular Dichroism Flashcards
What is Circular dichroism?
A technique used to work out the secondary structure
- absorption spectroscopy method based on the differential absorption of left and right circularly polarised light by chiral molecules
How can you measure the peptide backbone of the protein?
- using far UV CD
- secondary structures will show up on graphs
What are the different characteristics for secondary structures present on a CD spectrum?
Alpha helix - characteristics of the protein if present:
trough of 208nm, 222nm & peak at 190nm
Beta Sheet - characteristics of the protein if present
Trough at 216 nm & peak at 200 nm
Random Coil-
When protein hasn’t folded correctly
What else other than secondary structures can be seen from a CD spectrum?
CD signals from side chains that absorb light
- tryptophan, tyrosine, phenylalanine
Will show a change has occurred but not what has happened
What is the difference between Far and Near UV?
Near UV has a higher wavelength
- signals are smaller in Near UV
Far UV only needs a small concentration to work, where as Near UV a large concentration is needed.
What is ellipticitiy?
Elliptically polarised light is light that is not fully circular polarised, but instead is elliptical in shape
What does mean residue ellipticity show?
Works out per residue how much CD is present
How can linear polarised light turn into circular polarised light?
CD instrumentation
- photoelastic modulator (PEM) is used that turns linear light into left and right circular polarised light sequentially
- Detector detects the light from PEM
How is β2-microglobulin associated with amyloids?
Beta 2 - microglobulin Is associated with amyloid plaque formation
- Amyloid plaque = formation of beta helix formation (large fibre complex)
What are amyloids?
- v.high in β-sheets
- forms fibres
- associated with numerous diseases such as Alzheimer’s and prions
What is hemodialysis-related amyloidosis?
repeated hemodialysis causes β2-microglobulin deposits in tissues
- leads to excessive bone growth
- carpal tunnel syndrome
What is the molecular basis of diseases caused by β2-microglobulin?
- Native β2-microglobulin will undergo complete unfolding into random coil-β
- It will then refold into a beta-helix formation
How does using CD spectra help determine the molecular basis of diseases caused by β2-microglobulin?
CD spectra of β2M show a native β2M and then the spectra when several changes have occurred. When heated, the signal produced is similar to the native one.
When denatured, the signal is different showing mis-folding
Also can see mature and immature fibrils formation
- can also observe the kinetics of amyloid formation in real time
Which two mutations alter the severity of amyloid formation and how?
D59P - destabilises the core structure of the protein (increases and the likelihood the misfolding the protein)
- pushes β2-microglobulin to unfold
W60G - stabilises the protein
- pushes β2-microglobulin to return to original conformation rather than misfolding
What technique was used to asses how the two mutations altered amyloid formation?
Thermal denaturation by CD
