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Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes

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Book cover Bacterial Protein Secretion Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1615))

Abstract

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.

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Acknowledgements

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.

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Authors and Affiliations

  1. Laboratoire de Microbiologie et de Génétique Moléculaires, Centre de Biologie Intégrative (CBI), Université de Toulouse, CNRS, UPS, Toulouse, France

    Raffaele Ieva

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  1. Raffaele Ieva
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Correspondence to Raffaele Ieva .

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Editors and Affiliations

  1. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Laure Journet

  2. Institut de Microbiologie de la Méditerranée, Aix-Marseille Univ - CNRS, Marseille, France

    Eric Cascales

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Ieva, R. (2017). Site-Directed and Time-Resolved Photocrosslinking in Cells Metabolically Labeled with Radioisotopes. In: Journet, L., Cascales, E. (eds) Bacterial Protein Secretion Systems. Methods in Molecular Biology, vol 1615. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7033-9_19

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  • DOI: https://doi.org/10.1007/978-1-4939-7033-9_19

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7031-5

  • Online ISBN: 978-1-4939-7033-9

  • eBook Packages: Springer Protocols

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