Mapping of protein-peptide and protein-protein interactions by means of genetically encoded photocrosslinkers

• Applicant: Professorin Dr. Irene Coin
• Subject Area: Biochemistry
• Term: from 2013 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 236346005

• Final Report Year: 2020

Final Report Abstract

As major scientific achievements: we have revealed the mechanism of agonism and antagonism at the CRF1R; we have established a robust method to determine intermolecular pairs of proximal amino acids not only in ligand-receptor interfaces but in general in protein-protein interaction interfaces; we have developed a new system for the incorporation of Pyl-like ncAAs in mammalian cells and a general method for quantitative labeling of GPCRs on the surface of live cells. In this way, aims of project A and B of the original proposal are achieved. Although project C developed in a direction that was not planned at the beginning, it led to important results and nice publications. On these bases, projects involving 1) the study of ligand-GPCR interactions at other GPCRs, 2) the study of arrestin-GPCR interactions and 3) the generation of minimal sized FRET sensors for studies of receptor dynamics are ongoing and have a very promising long-term perspective.

Publications

• Incorporation of Unnatural Amino Acids into Proteins Expressed in Mammalian Cells. Methods Enzymol. 2016, 580, 89-107
  Serfling, R. & Coin, I.
  
• Designer tRNAs for efficient incorporation of non-canonical amino acids by the pyrrolysine system in mammalian cells. Nucleic Acids Res. 2018, 46 (1), 1-10
  Serfling, Robert; Lorenz, Christian; Etzel, Maja; Schicht, Gerda; Böttke, Thore; Mörl, Mario & Coin, Irene
  
• Structural insight into the activation of a class B G-protein-coupled receptor by peptide hormones in live human cells. Elife 2017, 6
  Seidel, Lisa; Zarzycka, Barbara; Zaidi, Saheem A; Katritch, Vsevolod & Coin, Irene
  
• Application of non-canonical crosslinking amino acids to study protein-protein interactions in live cells. Curr. Opin. Chem. Biol. 2018, 46, 156-163
  Coin, Irene
  
• Mapping of Protein Interfaces in Live Cells Using Genetically Encoded Crosslinkers. Methods Mol. Biol. 2018, 1728, 221-235
  Seidel, Lisa & Coin, Irene
  
• Optimizing the genetic incorporation of chemical probes into GPCRs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells. J Vis Exp 2018, (134)
  Serfling, Robert; Seidel, Lisa; Böttke, Thore & Coin, Irene
  
• Exploring pairwise chemical crosslinking to study peptide-receptor interactions. ChemBioChem 2019, 20 (5), 683-692
  Seidel, Lisa; Zarzycka, Barbara; Katritch, Vsevolod & Coin, Irene
  
• Quantitative single-residue bioorthogonal labeling of G protein-coupled receptors in live cells. ACS Chem Biol 2019, 14 (6), 1141-1149
  Serfling, Robert; Seidel, Lisa; Bock, Andreas; Lohse, Martin J.; Annibale, Paolo & Coin, Irene