SFP11: Protein Structure Determination (not Finished) Flashcards
What is circular dichroism?
An optical method to measure secondary structure content of proteins, fractional contributions from different secondary structure elements can be analysed
Synchrotron radiation is needed to obtain information below 190nm
What is Fourier-transform infrared (FTIR) spectroscopy?
Chemical bonds vibrate with frequencies in the IR range
Amide and carbonyl vibrations are affected by hydrogen bonding
Secondary structure elements shift and bind to characteristic frequencies
IR spectra are reported in wave numbers = 1/lambda (cm-1)
How does FTIR reveal protein secondary structure?
Secondary structure content of proteins can be determined from Amide I infrared band;
Spectral deconvolution allows quantitative determination of relative contributions from helix, sheet and random coil
What is an advantage of FTIR?
FTIR can be used to study membrane proteins
What is a disadvantage of FTIR?
Samples must be dehydrated to minimise absorption by water
What can be used to study hydrated samples?
Raman spectroscopy
What is analytical ultracentrifugation used for?
AUC can be used to estimate protein size and allows proteins complexes to be configured in vitro
How does analytical ultracentrifugation work?
Protein is removed from solution by ultracentrifugation
Measures sedimentation velocity
- smaller proteins sediment more slowly
- larger complexes sediment faster
Measures protein diffusion (how broad is the sedimentation boundary):
- smaller proteins diffused faster larger complexes diffuse more slowly
What are hydrodynamic methods - AUC
Shclieren optics and fluorescence allow monitoring protein sedimentation in real time
Name methods for protein structure determination
1) X-ray cystallography
2) nuclear magnetic resonance spectroscopy (NMR)
3) cryo-electron microscopy - membrane proteins can be studied
4) electron microscopy and electron tomography - overall shape can be obtained from electron micrographs
What is the process of X-ray crystallography?
- Crystallisation of the purified recombinant protein
- X-ray diffraction (typically using synchrotron source, high intensity, tuneable wave)
- Obtaining phases, usually incorporating heavy elements
- Obtaining electron density maps
- Computational determination of structure (fitting the protein chain)
- Optimisation of calculated structure
Why do we use X-Rays?
Their wavelength is around 1.54 angstroms
What is the Bragg law?
nλ = d . sin(θ)
Where lamda = wavelength
D= intermolecular distance
Theta= scattering angle
What does Bragg Law relate to?
Electrons within the protein scatter C-rays constructively
