• Home
  • Search Results
  • Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

Extending the cross-linking/mass spectrometry strategy: Facile incorporation of photo-activatable amino acids into the model protein calmodulin in Escherichia coli cells.

Methods (San Diego, Calif.) (2015-03-03)
Christine Piotrowski, Christian H Ihling, Andrea Sinz
ABSTRACT

Photo-induced cross-linking is a highly promising technique to investigate protein conformations and protein-protein interactions in their natural cellular environment. One strategy relies on the non-directed incorporation of diazirine-containing photo-activatable amino acids into proteins and a subsequent cross-link formation induced by UV-A irradiation. The advantage of this photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can also be targeted, which is advantageous for investigating membrane proteins. Here, we present a simplified protocol that relies on the use of mineral salts medium without any special requirements for the incorporation of photo-methionines into proteins in Escherichia coli cells. The possibility to perform these experiments in E. coli is especially valuable as it is the major system for recombinant protein production. The method is exemplified for the Ca(2+) regulating protein calmodulin containing nine methionines, which were found to be replaced by their photo-activatable analogues. Our protocol allows the facile and stochastic incorporation of photo-methionines as the basis for conducting photo-cross-linking experiments in E. coli in an efficient manner.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Sigma-Aldrich
Acetonitrile, ACS reagent, ≥99.5%
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) trifluoroacetic acid, suitable for HPLC
Sigma-Aldrich
Acetonitrile solution, contains 0.1 % (v/v) formic acid, suitable for HPLC
Sigma-Aldrich
Acetonitrile, puriss. p.a., ACS reagent, reag. Ph. Eur., ≥99.5% (GC)
Sigma-Aldrich
Acetonitrile, biotech. grade, ≥99.93%
Millipore
Acetonitrile solution, suitable for HPLC, acetonitrile:water 5:95% (v/v), 10 mM Ammoniumbicarbonate, pH 10,0
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
Acetonitrile, ≥99.5% (GC)
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile solution, contains 0.05 % (v/v) trifluoroacetic acid
Sigma-Aldrich
Acetonitrile, for preparative HPLC, ≥99.8% (GC)
Sigma-Aldrich
Acetonitrile
Sigma-Aldrich
Acetonitrile, for DNA synthesis
Sigma-Aldrich
Acetonitrile, configured for PerkinElmer 8900, configured for Polygen, for DNA synthesis
Sigma-Aldrich
Acetonitrile, for DNA synthesis
Sigma-Aldrich
Ultrapure Acetonitrile