Proteins

Question 0

Anfinsen 1950s

Answer 0

Ribonuclease - single polypeptide chain with 4 disulphides bridges
Urea disrupts non-covalent bonds 8M
B-metacaptoethanol reversibly cleaves disulphides bonds, in excess converts disulphides to sulfhydryls
Denature to random coil
If urea and B-metcaptoethanol removed by dialysis, ribonuclease regains enzymatic activity
Information to specify the catalytically active structure is contained in aa sequence
- remove urea first –> fully functioning
- remove B-metcaotoethanol first –> 1% functioning
Add traces, catalysed rearrangement, as decrease in free energy as converted to stable conformation

Question 1

Frederick Sanger

Answer 1

Determined aa sequence of insulin

Protein has a precisely defined aa sequence, unique

Question 2

Edman degradation

Answer 2

Remove one amino acid at a time form a polypeptide
Cyclic derivative of terminal amino acid identified by chromatograph and elution time from a separating column that is compared to a data base

Question 3

Mass spectrometry

Answer 3

Primary amino acid sequence

Protein fragments ionised, compared to a database

Question 4

X-Ray crystallography

Answer 4

Precise 3D location of electrons
X-Rays are scattered and recombined depending on atomic configuration
Forms electron density map (contours) can be stacked –> 3D

Question 5

NMR

Answer 5

3D arrangements
Structure and dynamics in solution
Chemical shift and splitting patterns depending on environment

Question 6

ATCase

Answer 6

In e.coli shows all-or-nothing conformational change upon bindi of CTP - almost rey
Shown using x-Ray crystallography
Change from relaxed to tense state - movement of dimers and trimmers