Building and Breaking Bonds via a Compact S-Propargyl-Cysteine to Chemically Control Enzymes and Modify Proteins

Angew Chem Int Ed Engl. 2018 Sep 24;57(39):12702-12706. doi: 10.1002/anie.201806197. Epub 2018 Sep 5.

Abstract

Analogous to reversible post-translational protein modifications, the ability to attach and subsequently remove modifications on proteins would be valuable for protein and biological research. Although bioorthogonal functionalities have been developed to conjugate or cleave protein modifications, they are introduced into proteins on separate residues and often with bulky side chains, limiting their use to one type of control and primarily protein surface. Here we achieved dual control on one residue by genetically encoding S-propargyl-cysteine (SprC), which has bioorthogonal alkyne and propargyl groups in a compact structure, permitting usage in protein interior in addition to surface. We demonstrated its incorporation at the dimer interface of glutathione transferase for in vivo crosslinking via thiol-yne click chemistry, and at the active site of human rhinovirus 3C protease for masking and then turning on enzyme activity via Pd-cleavage of SprC into Cys. In addition, we installed biotin onto EGFP via Sonogashira coupling of SprC and then tracelessly removed it via Pd cleavage. SprC is small in size, commercially available, nontoxic, and allows for bond building and breaking on a single residue. Genetically encoded SprC will be valuable for chemically controlling proteins with an essential Cys and for reversible protein modifications.

Keywords: Sonogashira coupling; palladium-mediated cleavage; propargyl cysteine; reversible protein modification; thiol-yne.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Archaeal Proteins / chemistry
  • Archaeal Proteins / genetics
  • Archaeal Proteins / metabolism
  • Biotin / chemistry
  • Catalysis
  • Catalytic Domain
  • Click Chemistry
  • Cysteine / chemistry*
  • Cysteine / metabolism
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / metabolism*
  • Enterovirus / enzymology
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Methanosarcina / metabolism
  • Mutagenesis, Site-Directed
  • Palladium / chemistry
  • Pargyline / chemistry
  • Thioredoxins / chemistry
  • Thioredoxins / genetics
  • Thioredoxins / metabolism
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism*

Substances

  • Archaeal Proteins
  • Viral Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Thioredoxins
  • Palladium
  • Biotin
  • Pargyline
  • Cysteine Endopeptidases
  • 3C proteases
  • Cysteine

Supplementary concepts

  • Rhinovirus B