lecture 9- Circular dichroism spec Flashcards
Circular dichroism occurs
when a molecule differentially absorbs left and right circularly polarised light
Light produced by natural sources?
Unpolarised
Unpolarised light waves are randomly
orientated around the beam axis
passing light through a filter will select for ?
Just one of those directions
Polarisation filters are made of?
are made of long chains of organic molecules, arranged in parallel to each other.
When light passes through the filter, the filter absorbs?
components of electric fields that are parallel to the direction in which the organic molecules are arranged.
polarised?
light that comes out would have its electric field oscillating along one direction
Reflection also causes
a partial linear polarization in the direction parallel to the reflected surface.
What light is polarised in polaroid sunglasses?
light coming from glares off the roads or from a water surface is polarized
Sunglasses (polaroid) have what type of filter?
Polarising filter
Glares when wearing polaroid sunglasses
A large portion of the light produced by a glare is unable to travel through the sunglasses
Light can be polarised by which two ways?
Linearly or circularly polarised
All polarised light states can be described by?
sumoftwolinearlypolarisedstatesatrightanglestoeach other
How can we get Circularly polarised light from the sum of two linearly polarised states?
using a second filter, causes quarter wave retardation
The quarter-wave retardation plate is made of?
a sheet of birefringent (double refracting) material
Thickness of the polarised light entering the phase such that ?- circularly polarised
- Horizontally and vertically polarized light entering in phase will emerge from the retardation plate 1/4 of a wavelength out of phase.
Circularly polarised light can be ?
right or left handed
how do you get plane polarised light?
When you add together left-handed and right-handed circularly polarised light
if a sample absorbs one more that the other, what kind of light do you get
elliptically polarised light.
L and R beams have different amplitudes
What properties must a molecule have in order to create 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
Chiral molecules have pairs of ??
Enantiomers - non super impossible mirror images
D AND L
Light source for CD spec?
U.V.
purpose of a photo elastic modulator and what it actually is?
normally a quartz crystal subjected to an oscillating electric field.
to form left and right polarised light- plane polarised light
Frequency of the light passing though the photo elastic modulator ?
50Hz
what is kept constant in. a CD spec kit
light intensity
What is detected by a photomultiplier ?
differential absorption of left and right polarised light
When light is detected by a photomultiplier??- units
converted into ellipticity (units millidegrees)
ellipticity
is the unit of circular dichroism and is defined as the tangent of the ratio of the minor to major elliptical axis
ΔA
circular dichroism
uv light causes?
electronic transitions at a particular wavelength
if a molecule contains a chiral chromophore ? the CD signal will be ?
Non zerp
CD=?
= ΔA(λ) = A(λ)Left CPL - A(λ)Right CPL
Cpl?
component
positive CD signal?
left CPL is absorbed more than right CPL
Negative CD signal?
If right CPL is absorbed more than left CPL
CD spectrum may exhibit what type of peaks?
pos and neg
θ
ellipticity
Interconversion between θ and ΔA
ΔA=θ/32.982
Looking at CD spec what are we looking at ?
difference in left and right polarised light
To increase accuracy of SD spec?
increase data points eg every 0.2 nm
CD x and y plot
CD (y) vs wavelength (x)
Uses of CD spectroscopy
- Protein secondary structure determination
- Monitoring changes in protein structure
- Ligand binding
- Protein folding / unfolding
- Effects of environment on protein structure - Nucleic acid structure
- Protein-protein and protein-DNA interactions
which two chromophores found In proteins can be utilised to determine protein secondary structure
Far uv spec:
bellow 240 nm
peptide
sensitive to changes in secondary structure
Near UV:
above 240nm
chromophore: tryptophan, DNA
festive to overall conformation
How are unknown protein secondary structures determined?
using known
standard curves
associated with different secondary structures
CD analysis tool?
dichroweb
pros of CD secondary structure analysis?
- Simple and quick, no extensive sample preparation
- Fairly reliabe
- obtainable for a wide range of proteins
- low sample conc in aq solution
disadvantages of CD secondary structure analysis
- Must have an accurate measure of your protein concentration
- Dependent on comparison
- CD spec also dependent on tertiary structure too
- Beta determination are harder as they are more variable
- strength of short sections not as good
Monitoring changes in protein structure examples
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
GCN4-p1 example of monitoring changes in secondary structure ?
100% helical at 0 degrees
melts to a random coil at high temps
TFE?
example of a solvent which causes a coiled coil to become a single stranded alpha helix
nucleotides are chiral due to?
pentose ring
what kind of CD spectra of nucleotides?
albeit weak CD spectra
with increasing structural order and asymmetry of nucleotides
Increasing strength of CD spec
A and B form of DNA give?
different CD spec
when proteins and nucleic acids bind to one and other?
changes in secondary structure which can be observed by CD
to observe changes in protein secondary structure following binding to DNA what uv used
far uv
bellow 240nm
to observe changes in DNA secondary structure following binding to protein
Near UV
above 240nm
