Download Stabilization of Low Affinity Protein-Protein Interactions by

25th Croatian meeting of chemists and chemical engineers, Poreč, 2017.
Stabilization of Low Affinity Protein-Protein Interactions by Site-Specific
Incorporation of Unnatural Amino Acids
Stabilizacija proteinskih interakcija niskog afiniteta pomoću mjesnospecifične ugradnje neprirodnih aminokiselina
…
Marko Cigler,1 Kathrin Lang1
1
Technical University Munich – Institute for Advanced Studies, Garching, Germany
...
E-mail: [email protected]
…
The introduction of new chemical functionalities into proteins represents a promising
approach for investigating and manipulating diverse biological processes. Among a number
of different approaches, the expansion of the genetic code has emerged as an eminent tool
for in vivo site-specific incorporation of unnatural amino acids (UAAs) into proteins [1]. The
requirements for genetic code expansion include an orthogonal aminoacyl-tRNA synthetase
(aaRS), engineered to specifically transfer the UAA, but no natural amino acids, onto its
orthogonal tRNA. Being a specific substrate only to the orthogonal aaRS, an orthogonal tRNA
is able to direct the incorporation of the UAA during translation in response to a unique
codon, most commonly the amber stop codon, in the gene of interest.
Over the past decade, a plethora of UAAs bearing different functional groups have
successfully been incorporated into proteins, both in bacteria as well as in eukaryotic cells,
giving new insights into their structures and functions. The development of fast,
chemoselective and high-yielding bioorthogonal reactions between UAAs and externally
added chemical probes, for example, offers a unique opportunity for imaging individual
proteins and labeling proteomes [1c, 2]. Their high reactivity, however, makes them unfit for
investigating protein-protein interactions in their native conformations.
For this purpose, we are exploring bioreactive bromoalkyl-bearing UAAs that are inert
under physiological conditions but react with nucleophilic natural amino acids such as
cysteines in a proximity enhanced manner [3]. By synthesizing and incorporating the
electrophilic UAAs site-specifically into proteins of interest, we were able to use these
reactions in vivo to covalently link positions that are brought into close proximity by the
formation of the protein complex. We applied this method for chemical stabilization and
crystallization of low affinity protein complexes in their native conformations which has,
until now, been very challenging.
References
[1] (a) C. C. Liu, P. G. Schultz, Annu. Rev. Biochem. 79 (2010) 413-444. (b) L. Davis, J. W. Chin, Nat.
Rev. Mol. Cell. Biol. 13 (2012) 168-182. (c) K. Lang, J. W. Chin., Chem. Rev. 114 (2014) 4764-4806. (d)
J. W. Chin, Ann. Rev. Biochem. 83 (2014) 379-408.
[2] (a) J. A. Prescher, C. R. Bertozzi, Nat. Chem. Biol. 1 (2015) 13-21. (b) E. M. Sletten, C. R. Bertozzi,
Angew. Chem. Int. Ed. 48 (2009) 6974-6998. (c) K. Lang, J. W. Chin., ACS Chem. Biol. 9 (2014) 16-20.
[3] X-H. Chen, Z. Xiang, Y. S. Hu, V. K. Lacey, H. Cang, L. Wang, ACS Chem. Biol. 15 (2014) 1956-1961.