Chem 135

Lecture 21

How would YOU incorporate an unnatural amino acid?

Chem 135

Lecture 21

Unnatural Amino Acids key players in Chemical Biology

Chem 135

Dave Tirrell Cal Tech residue specific incorporation

Peter Schultz the Scripps Institute site specific

(started at UC Berkeley!)

Lecture 21

Jason Chin Medical Research Council Lab of Molecular

Biology (Cambridge) site specific in organisms
(was post doc with Schultz)

Residue-specific amino acid incorporation (Tirrell method)

Chem 135

Lecture 21

Residue-specific incorporation of unnatural amino acids

Chem 135

Lang and Chin, Chemical Reviews, 2014, 114, 4764-4806.

Lecture 21

Unnatural amino acids incorporated using residue-specific methods

Chem 135

black = native RS / natural amino acid

blue = native RS / unnatural amino acid
green = extra native RS / unnatural amino acid
red = mutated RS / unnatural amino acid

Ngo and Tirrell, Accounts of Chemical Research, 2011, 44, 677-685

Lecture 21

Requirements for residue-specific amino acid incorporation

Chem 135

-unnatural amino acid must resemble the natural amino acid so that the tRNA
synthetase (RS) can charge it to the tRNA
-the amino acid being replaced must not be synthesized by the organism/cell
-the unnatural amino acid must be able to get into the cell
-uses the native codon
-uses the native tRNA
-often-times uses the native tRNA synthetase (RS)

Lecture 21

Site-specific Amino Acid Incorporation (the Schultz method)

Chem 135

also called amber suppression

Lecture 21

Site-specific incorporation
Chem 135

Get to know your STOP codons:

UAG = amber, named for friend (Harris Bernstein) of the discoverers.
Bernstein means amber in German.
UAA = ochre, a play on the color theme (most common in e coli, 2nd in
humans)
UGA = opal or umber (2nd most common in e coli, 1st in humans)

Lang and Chin, Chemical Reviews, 2014, 114, 4764-4806.

Lecture 21

Unnatural Amino Acids by site-specific incorporation

Chem 135

Lecture 21

Liu and Schultz, Annu. Rev. Biochem., 2010, 79, 413-44.

Requirements for site-specific incorporation of amino acids

Chem 135

-need a blank codon (amber stop codon)

-need an amber suppressor
-need orthogonal *tRNA/*RS pairs
-*tRNA must not be processed by native RSs
-*RS must charge *tRNA with the *AA
-*RS must NOT charge native tRNAs with *AA
-unnatural amino acid must enter cell
-unnatural amino acid does not necessarily look much like a
native amino acid
how do you design this? answer: you dont!

Lecture 21

A close look at orthogonality

Chem 135

Lang and Chin, Chemical Reviews, 2014, 114, 4764-4806.

Lecture 21

A close look at orthogonality

Chem 135

Lang and Chin, Chemical Reviews, 2014, 114, 4764-4806.

Lecture 21

Selection strategies for orthogonal tRNA/RS pairs

Chem 135

Lang and Chin, Chemical Reviews, 2014, 114, 4764-4806.

Lecture 21

Site-specific incorporation of unnatural amino acids

Chem 135

Lecture 21