L1 - Protein Structure Flashcards
Protein Structure
What are the hydrophobic side chains?
Alanine Isoleucine Leucine Methionine Phenylalaine Valine Proline Glycine
Name the 3 ionic interactions
Ionic
Van der Wall
Hydrogen
How to predict primary structures?
- From DNA sequences
- Amino acid sequencing - Edman degradation
- Mass spectrometry
What is Edman degradation?
Each round you discover the identity of a single amino acid.
In acidic conditions the terminal amino acid drops off and is analysed by high performance liquid chromatography.
How to predict secondary structures?
- From primary structure
- Protein databases
- Circular dichroism
What is circular dichroism for secondary structures?
CD spectroscopy in the far UV region reveals secondary structure.
All secondary structures hive characteristic shapes on the CD spectrum.
Fraction of each secondary structure present can be identified from the CD spectrum.
How to predict tertiary structures?
- Circular dichroism
- X-ray crystallography
- Nuclear magnetic resonance
- Electron microscopy
What is circular dichroism for tertiary structures?
CD spectroscopy in the near UV region reveals tertiary structure.
Absorbance of aromatic amino acids and disulphide bonds are affected by their local environment.
Protein unfolding can be followed by CD at different temperatures.
What is x-ray crystallography?
High energy, focussed beam of light is fired through a protein crystal.
A few x-rays are deflected giving rise to diffraction patterns.
Structure of protein is traced from diffraction pattern.
What is nuclear magnetic resonance?
In most atoms the spin of subatomic particles are paired against each other so no nucleus has no overall spin.
Some atoms have uneven numbers of protons and neutrons so the nuclei have a slight spin.
NMR active nuclei resonate at a specific frequency in a strong magnetic field.
Depending on local environment proteins resonate at slightly different frequencies - chemical shift
What is electron microscopy?
Uses negative stain or vitreous ice to preserve specimen.
Image analysis used to build up an average structure.
Price of all techniques
Solution biochem - very cheap Circular dichroism - cheap Crystallography - very expensive NMR - very expensive EM - fairly expensive
What is circular dichroism?
CD is the differential absorption of circularly polarised light
In NMR proteins are produced recombinantly by?
Usually in bacteria grown in media where the sole nutrient source is 15N and/or 13C, so that all protein produced are singly or doubly labelled with 15N and/or 13C in every atom
