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Photocrosslinking Approach to Investigate Protein Interactions in the BCL-2 Family

  • Jialing Lin
  • Arthur E. Johnson
  • Zhi Zhang
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

The Bcl-2 family of proteins regulates mitochondrial outer membrane permeability thereby making life or death decisions for cells. Most of Bcl-2 proteins contain hydrophobic regions that are embedded in intracellular membranes such as mitochondria. These membrane proteins are difficult to express and purify thereby preluding biochemical and biophysical characterizations. Here, we describe a photocrosslinking approach based on in vitro synthesis of Bcl-2 proteins with photoreactive amino acid analogs incorporated at specific locations. These photoreactive proteins are reconstituted into liposomal membranes with defined phospholipids or mitochondrial membranes isolated from animals, and their interactions with other Bcl-2 proteins are detected by photocrosslinking.

Key words

Apoptosis Mitochondria Bcl-2 Bax BH3-only proteins Photocrosslinking Membrane protein interaction Membrane permeabilization 

Notes

Acknowledgments

This work was supported by the United States National Institutes of Health grants (R01GM062964, and P20GM103640), Oklahoma Center for the Advancement of Science and Technology grant (HR16-026), and Presbyterian Health Foundation grant (GRF00000125) to J.L.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  2. 2.Stephenson Cancer CenterUniversity of Oklahoma Health Sciences CenterOklahoma CityUSA
  3. 3.ReCode Therapeutics, Inc.DallasUSA

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