Protein Structure and Analysis

Question 1

Structural Elements of Proteins

Answer 1

Primary structure (231 aa)
Secondary structure (1 alpha helix, 11 beta strands)
Tertiary can (beta can)
Quaternary structure (tetramer, homotetramer)

Question 2

Fluorescent Proteins

Answer 2

Located in the geometric centre of the molecule
Formed by the tripeptide Ser-Tyr-Gly
Autocatalytic reaction in presence of oxygen.
Genetic coding of colour and fluorescence
GFP is monomeric in solution

Question 3

Applications of GFP and derivatives

Answer 3

Fusion proteins are usually introduced into cells as DNA constructs
Cloning vector for expressing GFP fusion proteins in mammalian cells (constructed in bacteria)

Question 4

Purification of recombinant proteins

Answer 4

6 Histodines are called a histag
Have a metal affinity and bind to beads so purified using ion affinity chromatography

Question 5

Immobilised metal ion affinity chromatography

Answer 5

Alternative affinity chromatography methods available:
- GST tagging
- Immunoprecipitation
Protons separate protein from matrix

Question 6

Gel Filtration Chromatography

Answer 6

Gel beds separate molecules by size.
Optical detector, and the proteins are franctioned by size