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Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes

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Proteomics in Systems Biology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1394))

Abstract

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this “traditional” cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.

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Acknowledgements

This work is funded by the BMBF (Pronet-T3). We would like to thank Dr. Christian Ihling for LC/MS analyses, Dr. Knut Kölbel for fruitful discussions, and Dirk Tänzler for excellent technical support. Prof. Jens Meiler and Dr. David Nanneman, Vanderbilt University, are acknowledged for introducing P.L. into Rosetta, Prof. Gunter Fischer and Dr. Cordelia Schiene-Fischer, Max-Planck Forschungsstelle Halle, are acknowledged for generously providing their cell culture facilities. We are indebted to Prof. Mats Paulsson and Dr. Frank Zaucke; University of Cologne, for their help with laminin and nidogen expression and purification.

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Authors and Affiliations

  1. Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120, Halle/Saale, Germany

    Philip Lössl & Andrea Sinz

  2. Biomolecular Mass Spectrometry and Proteomics, Netherlands Proteomics Center, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands

    Philip Lössl

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  1. Philip Lössl
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  2. Andrea Sinz
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Correspondence to Andrea Sinz .

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Editors and Affiliations

  1. University of Regensburg Institute of Functional Genomics, Regensburg, Germany

    Jörg Reinders

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Lössl, P., Sinz, A. (2016). Combining Amine-Reactive Cross-Linkers and Photo-Reactive Amino Acids for 3D-Structure Analysis of Proteins and Protein Complexes. In: Reinders, J. (eds) Proteomics in Systems Biology. Methods in Molecular Biology, vol 1394. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3341-9_9

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  • DOI: https://doi.org/10.1007/978-1-4939-3341-9_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3339-6

  • Online ISBN: 978-1-4939-3341-9

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