Binding Specificities of Nanobody-Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry

Köhler, Martin and Neff, Christoph and Perez, Camilo and Brunner, Cyrill and Pardon, Els and Steyaert, Jan and Schneider, Gisbert and Locher, Kaspar P. and Zenobi, Renato. (2018) Binding Specificities of Nanobody-Membrane Protein Complexes Obtained from Chemical Cross-Linking and High-Mass MALDI Mass Spectrometry. Analytical chemistry, 90 (8). pp. 5306-5313.

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The application of nanobodies as binding partners for structure stabilization in protein X-ray crystallography is taking an increasingly important role in structural biology. However, the addition of nanobodies to the crystallization matrices might complicate the optimization of the crystallization process, which is why analytical techniques to screen and characterize suitable nanobodies are useful. Here, we show how chemical cross-linking combined with high-mass matrix-assisted laser/desorption ionization mass spectrometry can be employed as a fast screening technique to determine binding specificities of intact nanobody-membrane protein complexes. Titration series were performed to rank the binding affinity of the interacting nanobodies. To validate the mass spectrometry data, microscale thermophoresis was used, which showed binding affinities of the stronger binding nanobodies, in the low μM range. In addition, mass spectrometry provides access to the stoichiometry of the complexes formed, which enables the definition of conditions under which homogeneous complex states are present in solution. Conformational changes of the membrane protein were investigated and competitive binding experiments were used to delimit the interaction sites of the nanobodies, which is in agreement with crystal structures obtained. The results show the diversity of specifically binding nanobodies in terms of binding affinity, stoichiometry, and binding site, which illustrates the need for an analytical screening approach.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Perez)
UniBasel Contributors:Perez, Camilo
Item Type:Article, refereed
Article Subtype:Research Article
Note:Publication type according to Uni Basel Research Database: Journal article
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Last Modified:10 Apr 2020 16:35
Deposited On:10 Apr 2020 16:35

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