bi5001239_si_001.pdf (388.78 kB)
Structural Basis of Improved Second-Generation 3‑Nitro-tyrosine tRNA Synthetases
journal contribution
posted on 2015-12-17, 01:19 authored by Richard
B. Cooley, Jessica L. Feldman, Camden M. Driggers, Taylor A. Bundy, Audrey L. Stokes, P. Andrew Karplus, Ryan A. MehlGenetic code expansion has provided
the ability to site-specifically
incorporate a multitude of noncanonical amino acids (ncAAs) into proteins
for a wide variety of applications, but low ncAA incorporation efficiency
can hamper the utility of this powerful technology. When investigating
proteins containing the post-translational modification 3-nitro-tyrosine
(nitroTyr), we developed second-generation amino-acyl tRNA synthetases
(RS) that incorporate nitroTyr at efficiencies roughly an order of
magnitude greater than those previously reported and that advanced
our ability to elucidate the role of elevated cellular nitroTyr levels
in human disease (e.g., Franco, M. et al. Proc. Natl. Acad. Sci. U.S.A 2013, 110, E1102). Here, we explore the origins of the improvement achieved in these
second-generation RSs. Crystal structures of the most efficient of
these synthetases reveal the molecular basis for the enhanced efficiencies
observed in the second-generation nitroTyr-RSs. Although Tyr is not
detectably incorporated into proteins when expression media is supplemented
with 1 mM nitroTyr, a major difference between the first- and second-generation
RSs is that the second-generation RSs have an active site more compatible
with Tyr binding. This feature of the second-generation nitroTyr-RSs
appears to be the result of using less stringent criteria when selecting
from a library of mutants. The observation that a different selection
strategy performed on the same library of mutants produced nitroTyr-RSs
with dramatically improved efficiencies suggests the optimization
of established selection protocols could lead to notable improvements
in ncAA-RS efficiencies and thus the overall utility of this technology.