Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes
To efficiently transport proteins into and across cellular membranes, specialized transport machineries engage in recognition events with different domains of their client proteins, forming sequential intermediate complexes. The short-lived nature of these interactions poses a big challenge in the identification of the key factors involved in transport reactions and their mechanism of action. Site-directed photocrosslinking is a powerful method for the detection and accurate mapping of interacting protein domains. This chapter describes a protocol that combines site-directed photocrosslinking to metabolic labeling of proteins and lipids as a method to characterize, with temporal and spatial resolution, the interactions of a secretory protein as it transverses the bacterial envelope.
Key wordsSite-directed photocrosslinking Transient protein–protein interaction Protein–lipid interaction Protein secretion Escherichia coli Autotransporter BAM complex
This protocol was originally developed in the laboratory of Dr. Harris Bernstein (National Institutes of Health, Bethesda, MD, USA). Plasmid pDULE-pBpa was kindly provided by Dr. Peter Schultz (Scripps Research Institute, La Jolla, CA, USA). R.I. is supported by the CNRS-Inserm ATIP-Avenir program.