optimizing-genetic-incorporation-chemical-probes-into-gpcrs-for-photo

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

University of Leipzig

Understanding the topology of protein-protein interactions is a matter of fundamental importance in the biomedical field. Biophysical approaches such as X-ray crystallography and nuclear magnetic resonance can investigate in detail only isolated protein complexes that are reconstituted in an artificial environment. Alternative methods are needed to investigate protein interactions in a physiological context, as well as to characterize protein complexes that elude the direct structural characterization. We describe here a general strategy to investigate protein interactions at the molecular level directly in the live mammalian cell, which is based on the genetic incorporation of photo- and chemical crosslinking noncanonical amino acids. First a photo-crosslinking amino acid is used to map putative interaction surfaces and determine which positions of a protein come into proximity of an associated partner. In a second step, the subset of residues that belong to the binding interface are substituted with a chemical crosslinker that reacts selectively with proximal cysteines strategically placed in the interaction partner. This allows determining inter-molecular spatial constraints that provide the basis for building accurate molecular models. In this chapter, we illustrate the detailed application of this experimental strategy to unravel the binding modus of the 40-mer neuropeptide hormone Urocortin1 to its class B G-protein coupled receptor, the corticotropin releasing factor receptor type 1. The approach is in principle applicable to any protein complex independent of protein type and size, employs established techniques of noncanonical amino acid mutagenesis, and is feasible in any molecular biology laboratory.

Lisa Seidel

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