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BB10186 > lecture 9- Circular dichroism spec > Flashcards

lecture 9- Circular dichroism spec Flashcards

1
Q

Circular dichroism occurs

A

when a molecule differentially absorbs left and right circularly polarised light

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2
Q

Light produced by natural sources?

A

Unpolarised

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3
Q

Unpolarised light waves are randomly

A

orientated around the beam axis

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4
Q

passing light through a filter will select for ?

A

Just one of those directions

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5
Q

Polarisation filters are made of?

A

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

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6
Q

When light passes through the filter, the filter absorbs?

A

components of electric fields that are parallel to the direction in which the organic molecules are arranged.

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7
Q

polarised?

A

light that comes out would have its electric field oscillating along one direction

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8
Q

Reflection also causes

A

a partial linear polarization in the direction parallel to the reflected surface.

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9
Q

What light is polarised in polaroid sunglasses?

A

light coming from glares off the roads or from a water surface is polarized

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10
Q

Sunglasses (polaroid) have what type of filter?

A

Polarising filter

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11
Q

Glares when wearing polaroid sunglasses

A

A large portion of the light produced by a glare is unable to travel through the sunglasses

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12
Q

Light can be polarised by which two ways?

A

Linearly or circularly polarised

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13
Q

All polarised light states can be described by?

A

sumoftwolinearlypolarisedstatesatrightanglestoeach other

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14
Q

How can we get Circularly polarised light from the sum of two linearly polarised states?

A

using a second filter, causes quarter wave retardation

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15
Q

The quarter-wave retardation plate is made of?

A

a sheet of birefringent (double refracting) material

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16
Q

Thickness of the polarised light entering the phase such that ?- circularly polarised

A
  • Horizontally and vertically polarized light entering in phase will emerge from the retardation plate 1/4 of a wavelength out of phase.
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17
Q

Circularly polarised light can be ?

A

right or left handed

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18
Q

how do you get plane polarised light?

A

When you add together left-handed and right-handed circularly polarised light

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19
Q

if a sample absorbs one more that the other, what kind of light do you get

A

elliptically polarised light.

L and R beams have different amplitudes

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20
Q

What properties must a molecule have in order to create circular dichroism ?

A
  • 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
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21
Q

Chiral molecules have pairs of ??

A

Enantiomers - non super impossible mirror images

D AND L

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22
Q

Light source for CD spec?

A

U.V.

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23
Q

purpose of a photo elastic modulator and what it actually is?

A

normally a quartz crystal subjected to an oscillating electric field.

to form left and right polarised light- plane polarised light

24
Q

Frequency of the light passing though the photo elastic modulator ?

A

50Hz

25
Q

what is kept constant in. a CD spec kit

A

light intensity

26
Q

What is detected by a photomultiplier ?

A

differential absorption of left and right polarised light

27
Q

When light is detected by a photomultiplier??- units

A

converted into ellipticity (units millidegrees)

28
Q

ellipticity

A

is the unit of circular dichroism and is defined as the tangent of the ratio of the minor to major elliptical axis

29
Q

ΔA

A

circular dichroism

30
Q

uv light causes?

A

electronic transitions at a particular wavelength

31
Q

if a molecule contains a chiral chromophore ? the CD signal will be ?

A

Non zerp

32
Q

CD=?

A

= ΔA(λ) = A(λ)Left CPL - A(λ)Right CPL

33
Q

Cpl?

A

component

34
Q

positive CD signal?

A

left CPL is absorbed more than right CPL

35
Q

Negative CD signal?

A

If right CPL is absorbed more than left CPL

36
Q

CD spectrum may exhibit what type of peaks?

A

pos and neg

37
Q

θ

A

ellipticity

38
Q

Interconversion between θ and ΔA

A

ΔA=θ/32.982

39
Q

Looking at CD spec what are we looking at ?

A

difference in left and right polarised light

40
Q

To increase accuracy of SD spec?

A

increase data points eg every 0.2 nm

41
Q

CD x and y plot

A

CD (y) vs wavelength (x)

42
Q

Uses of CD spectroscopy

A
  1. Protein secondary structure determination
  2. Monitoring changes in protein structure
    - Ligand binding
    - Protein folding / unfolding
    - Effects of environment on protein structure
  3. Nucleic acid structure
  4. Protein-protein and protein-DNA interactions
43
Q

which two chromophores found In proteins can be utilised to determine protein secondary structure

A

Far uv spec:
bellow 240 nm
peptide
sensitive to changes in secondary structure

Near UV:
above 240nm

chromophore: tryptophan, DNA
festive to overall conformation

44
Q

How are unknown protein secondary structures determined?

A

using known
standard curves
associated with different secondary structures

45
Q

CD analysis tool?

A

dichroweb

46
Q

pros of CD secondary structure analysis?

A
  1. Simple and quick, no extensive sample preparation
  2. Fairly reliabe
  3. obtainable for a wide range of proteins
  4. low sample conc in aq solution
47
Q

disadvantages of CD secondary structure analysis

A
  1. Must have an accurate measure of your protein concentration
  2. Dependent on comparison
  3. CD spec also dependent on tertiary structure too
  4. Beta determination are harder as they are more variable
  5. strength of short sections not as good
48
Q

Monitoring changes in protein structure examples

A

Ligand binding
Protein folding / unfolding
Effects of environment on protein structure (temp / pH / denaturants
To check if a newly purified protein is correctly folded
To determine if a mutant protein folds in the same way as wild type
To analyse biopharmaceutical products to confirm that they are still in a correctly folded active conformation

49
Q

GCN4-p1 example of monitoring changes in secondary structure ?

A

100% helical at 0 degrees

melts to a random coil at high temps

50
Q

TFE?

A

example of a solvent which causes a coiled coil to become a single stranded alpha helix

51
Q

nucleotides are chiral due to?

A

pentose ring

52
Q

what kind of CD spectra of nucleotides?

A

albeit weak CD spectra

53
Q

with increasing structural order and asymmetry of nucleotides

A

Increasing strength of CD spec

54
Q

A and B form of DNA give?

A

different CD spec

55
Q

when proteins and nucleic acids bind to one and other?

A

changes in secondary structure which can be observed by CD

56
Q

to observe changes in protein secondary structure following binding to DNA what uv used

A

far uv

bellow 240nm

57
Q

to observe changes in DNA secondary structure following binding to protein

A

Near UV

above 240nm

BB10186 (6 decks)

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