Biophysical Techniques Flashcards
What is circular dichroism?
Technique which relies on the differential absorption of left or right polarised light by optically active (chiral) molecules which gives an indication of protein secondary structure
What is an optically active molecule?
A sample is optically active if it can rotate the plane of polarisation of a beam of light passing through it (chiral)
What apparatus are used for CD?
Use a monochromator which generates the beam of light and a photoelastic modulator which generates left and right polarised light alternatively by voltage stressing a crystal at high frequencies
What is the method to determine secondary structure by CD?
At any given wavelength (180nm-280nm) the absorption of left and right polarised light is conformation specific, dependent on the peptide bonds. Can measure the differential absorption by proteins. Some secondary structures produce characteristic CD spectra which can be compared to the sample.
What are the advantages of CD?
The process is very quick (30 minutes)
Only a small amount of material is needed (<100ug)
What are the disadvantages of CD?
If there is a mixture of different structures present, secondary structure will be hard to determine as only an average is given.
Only gives 2ndry structure information.
Limits what buffers you can use as buffers with chloride cause scattering.
What are questions that can be answered with CD?
To determine whether an expressed, purified protein is folded or if a mutation affects its conformation.
Can also look at B-DNA and A-DNA and see if structure changes if transctiption factor is bound
What is the radius of gyration (RG)?
Used to describe particle size in scattering and hydrodynamic experiments. Related to the radius of the protein and recording this can give an indication of the size and shape of the protein.
What is the background to scattering techniques such as MALS, SAXS and SANS?
When electromagnetic radiation is fired at a protein, scattering of the EM will depend on the wavelength of the EM compared to the Rg of the protein. If these are similar in size, scattering of the EM waves will cause interference dependent on the angle of scattering and the intensity of the recorded waves will be different; scattering intensity depends on the size and shape of the protein.
What is MALS?
Multiple Angle Light Scattering. Generally has three sensors surrounding a glass plate where protein is placed. EM is directed at the protein and sensors detect the intensity of the scattered waves.
The intensity at different angles will depend on the size and shape of the protein.
Why is MALS generally combined with SEC?
Size Exclusion Chromatography is also used in conjunction with MALS to give an estimate of the absolute molecular weight- produces a graph of time vs molecular mass
What are the advantages and disadvantages of MALS-SEC?
Advantages:
Is a callibration free technique; can detect aggregates at low concentrations
Disadvantages:
The molecular weight determinations are an estimate
High concentrations are problematic
Low throughput
What are SAXs and SANs?
SAX = Small Angle X-ray scattering SAN = Small Angle Neutron scattering
Describe the method of SAXs and what different results can tell us?
Fire X-rays at a purified sample; can record the diffuse scattering of X-rays; intensity of scattering is related to the protein in solution; can plot a graph of log-Intensity by angle
Large angles- give information about the particle shape due to interference
Small angles- give information about the molecular weight using the intercept and slope
Can do a Fourier transform of the graph to give information about maximum dimensions
Describe the method of SANs
Similar to SAXs but uses neutrons. Neutrons interact differently with different isotopes. Scattering by hydrogen atoms produces negative scattering but scattering by deuterium is positive.
By changing the ratios of H2O to D2O, can match solvent backgrounds to various macromolecular components of a solution- CONTRAST MATCHING
