Who discovered the first protein structure?
Max Perutz, myoglobin
Why is protein crystallography important?
Important for drug design
Describe the process of X-ray crystallography
- Grow a crystal of the candidate protein
- Mount the crystal, collect and evaluate preliminary diffraction data
- Complete data collection and estimate phases (calculate electron density map)
- Trace and refine chains
- Validate the structure
What does an electron density map look like?
Like a topographical map with all the lines and shit
At what X-ray crystallography can you determine individual atoms?
1.2 ANGSTROM
Give an example of a non-destructive method used to analyse structure and molecular dynamics of proteins
NMR
Which atoms does NMR use? (in biochemistry)
1H, 15N, 13C, 31P
H and P are highly abundant
C and N less so (
How does irradiation affect the atoms in NMR?
Causes the spin states to transition
What is the nuclear Overhauser effect?
The transfer of nuclear spin polarisation from one nucleus to another through space (rather than the spin-spincoupling effect which occurs through bonds).
This means atoms in close proximity to each other may cause a NOE.
What is NOESY?
Nuclear Overhauser enhancement spectroscopy:
A type of 2D NMR used in protein sequence NMR
What is HSQC?
Heteronuclear Single Quantum Coherence
2D NMR with one axis being a proton and the other being a heteronucleus (13C/15N).
Peaks for each unique proton attached to the heterologous nucleus.
Describe the principles of NMR
E.g. 1H NMR
- Strong magnet lines up the hydrogen nuclei
- nuclei may line up with spin up or spin down state
- Energy is added in the form of electromagnetic waves which cause the nuclei to spin flip to a higher energy state - Resonance condition
- The interaction between the radio waves/proton at is flips produces a peak on a graph
- The magnetic field strength is varied to produce spectra
- environment affects the protons - if covered by electromagnetic field then need to increase magnetic strength to cause resonance condition
What 2 other things may NMR be used for other than structural determination of proteins?
- Dynamic processes of proteins
- Monitoring of chemical reactions
Describe the process of mass spectrometry
- Molecules are ionised
- Electrical field accelerates ions
- Lightest ions arrive at detector first
What types of Mass spectrometry are there?
- MALDI
- ESI
- MALDI-TOF
What do the mass spectrometry graphs plot on the y and x axis?
Y - Intensity
X - Mass/charge ratio
