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

Methods : a Companion to Methods in Enzymology
Christine PiotrowskiAndrea 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...Continue Reading

Citations

Nov 11, 2015·Methods : a Companion to Methods in Enzymology·Carol E Parker, Christoph H Borchers
Aug 5, 2016·Microbial Biotechnology·Carlos G Acevedo-Rocha, Nediljko Budisa
Jul 28, 2020·The Journal of Physiology·N BraunS A Pless
Feb 23, 2017·Expert Review of Proteomics·Noa LipsteinOlaf Jahn
Oct 5, 2018·Frontiers in Molecular Neuroscience·Anne RehkampAndrea Sinz
Apr 22, 2018·Journal of the American Society for Mass Spectrometry·Claudio IacobucciAndrea Sinz
May 16, 2020·Journal of Proteome Research·Bastian KohlSebastian Hiller

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