lecture 9 Flashcards
What is circular dichroism?
Circular dichroism occurs when a molecule differentially absorbs left and right circularly polarised light
What is unpolarised light?
Light produced by natural sources.
Unpolarised light waves are randomly orientated around the beam axis.
What are polarisation filters made of?
long chains of organic molecules, arranged in parallel to each other.
What happens when light passes through a light filter?
the filter absorbs components of electric fields that are parallel to the direction in which the organic molecules are arranged.
Therefore, the light that comes out would have its electric field oscillating along one direction = polarised.
What is an example of polarised light
Polaroid sunglasses make use of polarizing filters. The light coming from glares off the roads or from a water surface is polarized.
How does polarising sunglasses work?
Since polaroid sunglasses have a polarizing filter, when you look at a glare through these sunglasses, a significant portion of the light coming from the “glare” is unable to travel through the sunglasses and reach your eyes.
What are the two components of linearly polarised light?
Electric vector direction constant
Magnitude varies
What are the two components of polarised light?
- Electric vector direction varies
- Magnitude constant
What are two differences between circular polarised light?
Circularly polarised light can be left or right polarised
What is circular dichroism?
L & R beams have different amplitudes
The resulting light is elliptically polarised
When does circular dichroism occur?
Circular dichroism occurs when a molecule differentially absorbs left and right circularly polarised light.
What are the properties in which the molecule can absorb circular dichroism?
Ability to absorb light (chromophore)
- Asymmetric / contain CHIRAL residues
- Contain a peptide bond
- Contain aromatic residues in asymmetric environments
- Contain DNA bases in asymmetric environments
What is chirality?
Chiral (handedness) molecules have pairs of optical isomers or enantiomers
Non-superimposable mirror images
Called L and D enantiomers
Why are we interested in chirality?
Each enantiomer of an optically active molecule interacts differently with left and right polarised light
How does circular dichroism interact with each different enantiomer?
L: Laevorotation (rotates to the left)
D: Dextrorotation (rotates to the right)
How does the equipment for CD spectroscopy expose the sample to left and right polarised light?
achieved by passing a beam of plane polarised light through a photoelastic modulator, which is normally a quartz crystal subjected to an oscillating electric field.
What does the output of the CD spectroscopy give us?
– the tangent of the ratio of the minor to major elliptical axis, units of millidegrees
Or ΔA (absorbance) / Δε (molar extinction coefficient).
How can we measure circular dichroism?
CD = ΔA(λ) = A(λ)Left CPL - A(λ)Right CPL
So if left CPL(component) is absorbed more than right CPL, the CD signal is positive
If right CPL is absorbed more than left CPL, the CD signal is negative.
How can we measure circular dichroism?
ellipticity
What is CD spectra an example of ?
- difference spectra
- We are looking at the difference between left and right polarised light
How can we increase the accuracy of CD spectra?
To increase accuracy, lots of data points are required.
- and each data point is read multiple times and averaged
What are the main uses of CD spectroscopy?
- Protein secondary structure - determination
- Monitoring changes in protein structure…
1) Ligand binding
2)Protein folding / unfolding
3)Effects of environment on protein structure
Nucleic acid structure
Protein-protein and protein-DNA interactions
How can we determine a proteins secondary structure?
- Known proteins were used to work out standard curves associated with different secondary structures
- unknown proteins can be compared to these
How Is a standard spectra determined?
- Best fitting procedures used
- there are Many different algorithms
- rely on using a number of CD spectra of proteins of known structure
What are the advantages of CD secondary structure analysis ?
- simple
- methods for analysing are reliable
- obtainable for a large range of proteins
- low sample concentration needed
- very quick
What are the disadvantages of CD secondary structure analysis?
- must have an accurate measure of protein conc
- depends on comparison
- the spectra of far UV VD depends on the tertiary structure too
- Beta structure are hard as they are more variable
- the strength for short sections of secondary structures Is not as good
