Lecture 5: Optical Activity and Fluorescence Flashcards
What is circular dichroism?
CD gives us values of molar ellipticity at a particular wavelength, which is the change in absorbance. It is written as [θ]λ.
ΔA = AL-AR = Δecl
- Change in e is small compared to e itself.
- CD has low sensitivity so we need a 100 microlitre sample at 10 micromolar.
- We can compare far-UV experimental CD curves with one generated from standard curves. This can show us the secondary structure content of a protein.
- CD can also be used to show secondary structure changes, based on changes in their CD spectra.0
- It can also show changes in DNA secondary structure (B and A DNA).
What is fluorescence?
Fluorescence arises from emission from an upper electronic state.
• Emission is normally at a lower wavelength than the absorbance.
• When radiation is absorbed, electrons move to a higher energy level.
• The electron then decays to the lowest energy level of the excited state, causing a lower wavelength emission.
• Measurement occurs by using two monochromators, one for excitation and one for emission
• Fluorescence is very useful because of its high level of sensitivity (nM and microlitres of sample can be used).
• It can be used on single molecules and even in vivo.
• It is used to probe a local environment and measure molecular dynamics in the ns range
How does fluorescent labelling work?
- Some molecules have intrinsic fluorescence (although not many). Examples include NADH, tryptophan and chlorophyll.
- Most molecules require extrinsic dyes or labelling via GFP. GFP has been modified to create many different colours.
- Another example of extrinsic dye is ethidium bromide for DNA.
- Fluorescence can be very sensitive to the environment.
- Some dyes can have special useful properties. For example, ANS only fluoresces when bound to exposed hydrophobic groups (e.g. in a denatured protein). Thermal denaturation can therefore be measured using these dyes.
What is fluorescence polarisation?
FP is a method to show movement of proteins.
Absorbance and emission depends on the direction of the transition dipole moment.
Polarised light will thus only excite a subset of fluorophores.
The output light will then also be polarised. If molecules move during the fluorescence then the output light will be depolarised.
We try to find a value called polarisation anisotropy (A).
A is defined as the degree of polarisation divided by the total fluorescence output. This can be measured using static or pulsed methods.
A= (I(parallel)-I(perpendicular))/(I(parallel)+2I(perpendicular))
Static depolarisation can be used in binding studies. When ligand is free, there is low A, it is unpolarised. When a ligand is bound, there is slow tumbling. There is an increase in fluorescence and polarisation on binding (high A).
What is Forster resonance energy transfer (FRET)?
FRET is a method which is used to measure the distance between two molecules.
Energy can be transferred when the emission spectrum of a donor and the excitation spectrum of an acceptor overlap.
Fluorescence intensities of donor and acceptor depend on spectral overlap and separation (r). We measure efficiency of transfer.
FI(d) is fluorescence intensity of the donor in the presence of the acceptor and FI(0) is the intensity in absence of the acceptor.
Another important measurement is R0 which is the Forster distance, where Et = 1/2.
E_t=(R_0^6)/(r^6+R_0^6 )
An example of FRET usage is in a GFP/BFP system. They are expressed together with a trypsin sensitive linker. When trypsin cleaves the linker, the proteins diffuse apart. We can also measure conformational changes. Another example of FRET being used is looking at the positioning of fluorescent sensors on a walking kinesin molecule.
What is fluorescence in situ hybridisation (FISH)?
Fluorescent probes are used to bind to nucleic acid sequences which are complementary.
• Fluorescently labelled probe DNA hybridises to denatured native DNA. This lights up chromosomes and allows them to be observed in a microscope.
• We can also look at mRNA or miRNA in cells.
How does real time quantitative PCR (qRT-PCR) work
This is a method to detect and quantify DNA or mRNA.
• An oligonucleotide probe is used with a fluorescent reporter dye at the 5’ end and a quencher at the 3’ end.
• The probes will hybridise to a PCR product. When intact, there is no fluorescence because of the quencher.
• During PCR, the 5’ nuclease activity of the polymerase cleaves the probe. This decouples the quenching, and fluorescence increases in each cycle.
What is fluorescence activated cell sorting (FACS)?
Sorts a mix of biological cells into two or more containers, one cell at a time based on fluorescent characteristics of each cell.
• It is an easy way to record fluorescent signals from each cell, as well as sorting them.
• The cell is given a charge before measurement. An electrostatic deflection system then sorts the cells.
