CH339K Proteins: Primary Structure, Purification, and Sequencing
All amino acids as incorporated are in the L-form • Some amino acids can be changed to D- after incorporation • D-amino acids occur in some non-protein molecules
A Modified Amino Acid That Can Kill You Histidine Diphthamide (2-Amino-3-[2-(3-carbamoyl-3-trimethylammonio-propyl)-3H-imidazol-4-yl]propanoate)
Diphthamide Continued – Elongation Factor 2 • Diphthamide is a modified Histidine residue in Eukaryotic Elongation Factor 2 • EF-2 is required for the translocation step in protein synthesis
Diphtheria Toxin Action Virus infects bacterium Infected bacxterium produces toxin Toxin binds receptor on cell Receptor-toxin complex is endocytosed Endocytic vessel becomes acidic Receptor releases toxin Toxin escapes endocytic vessel into cytoplasm Bad things happen
Diphtheria Toxin Action • Diphtheria toxin adds a bulky group to diphthamide • eEF2 is inactivated • Cell quits making protein • Cell(s) die • Victim dies
Polymerization DG0’ = +10-15 kJ/mol
In vivo, amino acids are activated by coupling to tRNA Polymerization of activated a.a.: DGo’ = -15-20 kJ/mol
In vitro, a starting amino acid can be coupled to a solid matrix • Another amino acid with • A protected amino group • An activating group at the carboxy group • Can be coupled • This method runs backwards from in vivo synthesis (C N)
Cis-trans isomerization in prolines • Other amino acids have a trans-cis ratio of ~ 1000:1 • Prolines have cis:trans ratio of ~ 3:1 • Ring structure of proline minimizes DG0 difference
Physical Methodsor How to Purify and Sequence a Weapons-Grade Protein
First Question How do I measure the amount of protein I have?
Beer-Lambert Law c = concentration l = path length e = extinction coefficient An Absorbance = 2 means that only 1% of the incident beam is getting through.
Transmittance and Absorbance Absorbance vs. Concentration Transmittance vs. Concentration
Second Question How can I spot my protein in the great mass of different proteins?
The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so: i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein