13 - Metabolic Labeling and Incorporation of Non-Natural Amino Acids

Question 1

metabolic labeling

Answer 1

• synthesis and modification are used to put synthetic tags into biomolecules
• natural molecular building block in media culture is replaced with a tagged chemical analog
• 2 step process

Question 2

click chemistry/bio-orthogonal chemistry

Answer 2

1. introduce a small unnatural handle (small modification)
2. react with a larger tag molecule to add functionality

• a catalyst is not needed as there the strain results in a high energy intermediate

Question 3

strain-promoted azide-alkyne cycloaddition

Answer 3

• treat a cell with a sugar that has an azide

- react with tags that react with azide

Question 4

Staudinger ligation

Answer 4

azide and phosphine ligation through copper catalyst

Question 5

zebrafish

Answer 5

• sugar with an azide is inserted into zebrafish embryos
• cycloalkyne that has a fluorophore is reacted with the azide
• fluorescence shows in what stage of development are the sugar and the alkynes are reacting

Question 6

SILAC

Answer 6

• cells grown in light and heavy medium
• cells are labelled and mixed together
• used on acrylamide gel then tryptic digestion
• mass spectroscopy shows how much a protein is affected by RNAi

Question 7

BONCAT

Answer 7

• uses AHA or HPG
• tRNA synthatase for Met is tricked by AHA or HPG
• modified amino acids are injected into mouse and are incorporated into proteins of the mouse normally
• treated with HPG results in an alkyne (AHA will result in azide)
• alkyne is reacted with fluorophore-labelled azide, catalyzed by copper
• polyacrylamide gel will show proteins in the mouse and that alkyne was incorporated

Question 8

metabolic labeling with photoreactive amino acids

Answer 8

• a diazirine agent is shined with UV light that results in carbene and nitrogen (by-product)
• carbene can form crosslinks to show PPI

Question 9

tRNA synthetase accepting an artifical amino acid

Answer 9

• site-mutagenesis is used to change the shape of the active site, allowing an analog of an amino acid to bind
• provides tissue specificity (where the mutant is expressed) where all proteins from that tissue will be labelled

Question 10

site-specific metabolic labelling

Answer 10

• find a tRNA and tRNA synthetase (charges tRNA with unnatural a.a.) for non-natural amino acid incorporation
• tRNA that was used binds to the UAG stop codon, but is changed to a non-natural amino acid
• synthetase engineered to have a binding site recognized only by the non-natural amino acid

Question 11

Professor Suga

Answer 11

• hypothesized that the first tRNA-synthetase was a ribozyme
• engineered a RNA-based tRNA synthetase
• resulted in Flexizyme

Question 12

flexizyme

Answer 12

• a ribozyme that charges particular tRNA with a specific a.a.
• resulted in amino-acylated tRNA

Question 13

amino-acylated tRNA

Answer 13

• allowed in vitro translation reactions to make proteins with unnatural a.a. easier
• result from flexizyme