Protein Structure

Question 1

Determine primary sequence of protein

Answer 1

Edman degradation -
PITC labels N-terminal aa
Cleave off using anhydrous acid
Identify using reverse-phase chromatography

OR
Tandem MS

Question 2

How to determine structure and Function from sequence

Answer 2

Sequence alignment with known proteins, and homology modelling
Secondary structure prediction
Recognition of motifs and domains
Signal sequence

Question 3

Conservation of protein structure

Answer 3

Type II RE’s have a conserved core of Asp co-ordinating Mg
Active site of enzymes
Actin and Hsp-70 conserved structure

Question 4

Convergent evolution

Answer 4

Chymotrypsin and subtilisin - proteases that use same catalytic mechanism
Conserved Asp-His-Ser catalytic residue
Different order in primary sequence, no structural homology,

Question 5

Foetal haemoglobin

Answer 5

2,3-BPG binds to T-state, stabilises T-state. Unbinds upon T-to-R transition.
H143S mutation of foetal haemoglobin lowers affinity for 2,3-BPG

Question 6

Bohr effect of haemoglobin

Answer 6

Acidic conditions stabilises T-state and decreases O2 binding affinity
Protonation forms a salt bridge within haemoglobin that stabilises T-state