In Vivo Site-Directed and Time-Resolved Photocrosslinking of Envelope Proteins

Abstract

In vivo site-directed photocrosslinking provides a means to probe the vicinity of proteins in their native cellular environment. Because this method relies on the incorporation of unnatural amino acid analogs that are similar in size to natural amino acids, crosslink products are indicative of direct protein-protein interactions. Here, we present the use of this approach to monitor both transient and stable interactions of two proteins of the envelope of Escherichia coli. First, we describe a protocol to characterize the interactions of a secretory protein as it transverses the bacterial envelope with temporal and spatial resolution. We combine site-directed photocrosslinking with radiolabeling of proteins and lipids. Second, we describe a method to purify a photocrosslinked partner protein and to analyze it by mass spectrometry. We use in-gel protein digestion and peptide fragmentation by MALDI-TOF/TOF tandem mass spectrometry to determine the site of interaction on the photocrosslinked partner.

Keywords: Bacterial envelope; Escherichia coli; Mass spectrometry; Protein secretion; Protein–lipid interaction; Protein–protein interaction; Site-directed photocrosslinking.