lecture 9

Question 1

What is circular dichroism?

Answer 1

Circular dichroism occurs when a molecule differentially absorbs left and right circularly polarised light

Question 2

What is unpolarised light?

Answer 2

Light produced by natural sources.

Unpolarised light waves are randomly orientated around the beam axis.

Question 3

What are polarisation filters made of?

Answer 3

long chains of organic molecules, arranged in parallel to each other.

Question 4

What happens when light passes through a light filter?

Answer 4

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.

Question 5

What is an example of polarised light

Answer 5

Polaroid sunglasses make use of polarizing filters. The light coming from glares off the roads or from a water surface is polarized.

Question 6

How does polarising sunglasses work?

Answer 6

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.

Question 7

What are the two components of linearly polarised light?

Answer 7

Electric vector direction constant

Magnitude varies

Question 8

What are the two components of polarised light?

Answer 8

• Electric vector direction varies

- Magnitude constant

Question 9

What are two differences between circular polarised light?

Answer 9

Circularly polarised light can be left or right polarised

Question 10

What is circular dichroism?

Answer 10

L & R beams have different amplitudes

The resulting light is elliptically polarised

Question 11

When does circular dichroism occur?

Answer 11

Circular dichroism occurs when a molecule differentially absorbs left and right circularly polarised light.

Question 12

What are the properties in which the molecule can absorb circular dichroism?

Answer 12

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

Question 13

What is chirality?

Answer 13

Chiral (handedness) molecules have pairs of optical isomers or enantiomers
Non-superimposable mirror images
Called L and D enantiomers

Question 14

Why are we interested in chirality?

Answer 14

Each enantiomer of an optically active molecule interacts differently with left and right polarised light

Question 15

How does circular dichroism interact with each different enantiomer?

Answer 15

L: Laevorotation (rotates to the left)
D: Dextrorotation (rotates to the right)

Question 16

How does the equipment for CD spectroscopy expose the sample to left and right polarised light?

Answer 16

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.

Question 17

What does the output of the CD spectroscopy give us?

Answer 17

– the tangent of the ratio of the minor to major elliptical axis, units of millidegrees
Or ΔA (absorbance) / Δε (molar extinction coefficient).

Question 18

How can we measure circular dichroism?

Answer 18

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.

Question 19

How can we measure circular dichroism?

Answer 19

ellipticity

Question 20

What is CD spectra an example of ?

Answer 20

• difference spectra

- We are looking at the difference between left and right polarised light

Question 21

How can we increase the accuracy of CD spectra?

Answer 21

To increase accuracy, lots of data points are required.

- and each data point is read multiple times and averaged

Question 22

What are the main uses of CD spectroscopy?

Answer 22

• 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

Question 23

How can we determine a proteins secondary structure?

Answer 23

• Known proteins were used to work out standard curves associated with different secondary structures
• unknown proteins can be compared to these

Question 24

How Is a standard spectra determined?

Answer 24

• Best fitting procedures used
• there are Many different algorithms
• rely on using a number of CD spectra of proteins of known structure

Question 25

What are the advantages of CD secondary structure analysis ?

Answer 25

• simple
• methods for analysing are reliable
• obtainable for a large range of proteins
• low sample concentration needed
• very quick

Question 26

What are the disadvantages of CD secondary structure analysis?

Answer 26

• 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