Proximity‐Triggered Covalent Stabilization of Low‐Affinity Protein Complexes In Vitro and In Vivo

Abstract

The characterization of low-affinity protein complexes is challenging due to their dynamic nature. Here, we present a method to stabilize transient protein complexes in vivo by generating a covalent and conformationally flexible bridge between the interaction partners. A highly active pyrrolysyl tRNA synthetase mutant directs the incorporation of unnatural amino acids bearing bromoalkyl moieties (BrCnK) into proteins. We demonstrate for the first time that low-affinity protein complexes between BrCnK-containing proteins and their binding partners can be stabilized in vivo in bacterial and mammalian cells. Using this approach, we determined the crystal structure of a transient GDP-bound complex between a small G-protein and its nucleotide exchange factor. We envision that this approach will prove valuable as a general tool for validating and characterizing protein-protein interactions in vitro and in vivo.

A methylated lysine is a switch point for conformational communication in the chaperone Hsp90

“…The same purification protocol was used for Rab1b, although the respective wash buffer was complemented with 0.2 mM PMSF, 0.01 mM GDP, cOmplete protease inhibitor cocktail tablets (Roche) for cell lysis (Rab1b wash buffer: 40 mM HEPES pH 8, 2 mM ß-ME, 1 mM MgCl 2 , 500 mM LiCl, 0.01 mM GDP, 20 mM imidazole; Rab1b elution buffer: 40 mM HEPES pH 8, 2 mM ß-ME, 1 mM MgCl 2 , 500 mM LiCl, 0.01 mM GDP, 300 mM imidazole). 85,87 The incorporation of met-ncbK and quantitative decaging to metK was confirmed using full-length ESI-LC MS (Phenomenex Jupiter TM C4 column (2 × 150 mm, 5 μm)) and carried out on an Agilent Technologies 1260 Infinity LC-MS system with a 6310 quadrupole spectrometer. Buffer A was 0.1% formic acid in water; buffer B was 0.1% formic acid in acetonitrile.…”

Expanding the Genetic Code to Study Protein–Protein Interactions

“…Proximity‐triggered protein crosslinking was in the beginning mainly used for intramolecular protein stapling, for the in vitro crosslinking of well‐established affibody complexes, and for the crosslinking of ligand–receptor pairs at the cell surface. Recently, its potential as a tool for in vivo trapping of biologically relevant protein–protein interactions, both in bacteria and mammalian cells, has been realized in diverse applications …”

“…By employing a highly efficient Mb PylRS mutant, a series of lysine derivatives bearing structurally flexible bromoalkyl chains of various lengths ( 17 , n =4–7) were site‐specifically incorporated into proteins both in E. coli and mammalian cells . Incorporation of 17 ( n =5 or 6) into sfGFP resulted in the formation of covalently linked sfGFP homodimer, with an ester bond between 17 in one sfGFP monomer and a specific glutamate residue in the other monomer.…”

Neue Mitglieder der Royal Society: P. L. Arnold, M. A. Brimble, F. Caruso, R. S. Goody, R. H. Crabtree, C. Bertozzi, J. Sauer / Jochen‐Block‐Preis: A. Vorholt / Otto‐Bayer‐Preis: T. Erb

“…Goodys Forschung dreht sich um GTPasen, einschließlich Rab‐GTPasen, und ihre Rolle in zellulären Transportprozessen. Er berichtete in der Angewandten Chemie über die kovalente Stabilisierung niederaffiner Proteinkomplexe …”