4. Methods

Question 1

Who discovered the first protein structure?

Answer 1

Max Perutz, myoglobin

Question 2

Why is protein crystallography important?

Answer 2

Important for drug design

Question 3

Describe the process of X-ray crystallography

Answer 3

1. Grow a crystal of the candidate protein
2. Mount the crystal, collect and evaluate preliminary diffraction data
3. Complete data collection and estimate phases (calculate electron density map)
4. Trace and refine chains
5. Validate the structure

Question 4

What does an electron density map look like?

Answer 4

Like a topographical map with all the lines and shit

Question 5

At what X-ray crystallography can you determine individual atoms?

Answer 5

1.2 ANGSTROM

Question 6

Give an example of a non-destructive method used to analyse structure and molecular dynamics of proteins

Answer 6

NMR

Question 7

Which atoms does NMR use? (in biochemistry)

Answer 7

1H, 15N, 13C, 31P

H and P are highly abundant
C and N less so (

Question 8

How does irradiation affect the atoms in NMR?

Answer 8

Causes the spin states to transition

Question 9

What is the nuclear Overhauser effect?

Answer 9

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.

Question 10

What is NOESY?

Answer 10

Nuclear Overhauser enhancement spectroscopy:

A type of 2D NMR used in protein sequence NMR

Question 11

What is HSQC?

Answer 11

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.

Question 12

Describe the principles of NMR

Answer 12

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

Question 13

What 2 other things may NMR be used for other than structural determination of proteins?

Answer 13

• Dynamic processes of proteins

- Monitoring of chemical reactions

Question 14

Describe the process of mass spectrometry

Answer 14

• Molecules are ionised
• Electrical field accelerates ions
• Lightest ions arrive at detector first

Question 15

What types of Mass spectrometry are there?

Answer 15

• MALDI
• ESI
• MALDI-TOF

Question 16

What do the mass spectrometry graphs plot on the y and x axis?

Answer 16

Y - Intensity

X - Mass/charge ratio