Abstract
In the cell, homo- and hetero-associations of polypeptide chains evolve and take place within subcellular compartments that are crowded with many other cellular macromolecules. In vivo chemical cross-linking of proteins is a powerful method to examine changes in protein oligomerization and protein-protein interactions upon cellular events such as signal transduction. This chapter is intended to provide a guide for the selection of cell membrane permeable cross-linkers, the optimization of in vivo cross-linking conditions, and the identification of specific cross-links in a cellular context where the frequency of random collisions is high. By combining the chemoselectivity of the homo-bifunctional cross-linker and the length of its spacer arm with knowledge on the protein structure, we show that selective cross-links can be introduced specifically on either the dimer or the hexamer form of the same polypeptide in vitro as well as in vivo, using the human type B nucleoside diphosphate kinase as a protein model.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Ji TH (1983) Bifunctional reagents. Methods Enzymol 91:580–609
Kluger R, Alagic A (2004) Chemical cross-linking and protein-protein interactions-a review with illustrative protocols. Bioorg Chem 32:451–472
Petrotchenko EV, Borchers CH (2010) Crosslinking combined with mass spectrometry for structural proteomics. Mass Spectrom Rev 29:862–876
Stengel F, Aebersold R, Robinson CV (2012) Joining forces: integrating proteomics and cross-linking with the mass spectrometry of intact complexes. Mol Cell Proteomics 11(R111):014027
Pornillos O, Ganser-Pornillos BK, Kelly BN et al (2009) X-ray structures of the hexameric building block of the HIV capsid. Cell 137:1282–1292
Mika JT, Poolman B (2011) Macromolecule diffusion and confinement in prokaryotic cells. Curr Opin Biotechnol 22:117–126
Wang K, Richards FM (1975) Reaction of dimethyl-3,3'-dithiobispropionimidate with intact human erythrocytes. Cross-linking of membrane proteins and hemoglobin. J Biol Chem 250:6622–6626
Bruce JE (2012) In vivo protein complex topologies: sights through a cross-linking lens. Proteomics 12:1565–1575
Boissan M, Lacombe ML (2011) Learning about the functions of NME/NM23: lessons from knockout mice to silencing strategies. Naunyn Schmiedebergs Arch Pharmacol 384:421–431
Lascu I, Gonin P (2000) The catalytic mechanism of nucleoside diphosphate kinases. J Bioenerg Biomembr 32:237–246
Dexheimer TS, Carey SS, Zuohe S et al (2009) NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III(1). Mol Cancer Ther 8:1363–1377
Postel EH, Berberich SJ, Flint SJ et al (1993) Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis. Science 261:478–480
Mesnildrey S, Agou F, Karlsson A et al (1998) Coupling between catalysis and oligomeric structure in NDP kinase. J Biol Chem 273:4436–4442
Karlsson A, Mesnildrey S, Xu Y et al (1996) Nucleoside diphosphate kinase. Investigation of the intersubunit contacts by site-directed mutagenesis and crystallography. J Biol Chem 271:19928–19934
Mesnildrey S, Agou F, Veron M (1997) The in vitro DNA-binding properties of NDP kinase are related to its oligomeric state. FEBS Lett 418:53–57
Agou F, Raveh S, Mesnildrey S et al (1999) Single strand DNA specificity analysis of human nucleoside diphosphate kinase B. J Biol Chem 274:19630–19638
Pogulis RJ, Vallejo AN, Pease LR (1996) In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol Biol 57:167–176
Kluger R (1997) Chemical cross-linking and protein function. In: Creighton TE (ed) Protein function a practical approach. IRL Press, Oxford, p 185
Morera S, Lacombe M-L, Xu Y et al (1995) X-Ray structure of nm23 Human Nucleoside Diphophate Kinase B complexed with GDP at 2A resolution. Structure 3:1307–1314
Agou F, Ye F, Goffinont S et al (2002) NEMO trimerizes through its coiled-coil C-terminal domain. J Biol Chem 277:17464–17475
Napetschnig J, Wu H (2013) Molecular Basis of NF-kappaB Signaling. Annu Rev Biophys 42:443–468
Acknowledgments
The authors are very grateful to Samuel Levy, MD for his critical reading of the manuscript. This work was supported, in whole or in part, by the Fondation ARC pour la recherche sur le cancer and La Ligue contre le cancer.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Agou, F., Véron, M. (2015). In Vivo Protein Cross-Linking. In: Meyerkord, C., Fu, H. (eds) Protein-Protein Interactions. Methods in Molecular Biology, vol 1278. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2425-7_26
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2425-7_26
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2424-0
Online ISBN: 978-1-4939-2425-7
eBook Packages: Springer Protocols