Martin Köhler1, Christoph Neff1, Camilo Perez2, Cyrill Brunner1, Els Pardon3, Jan Steyaert3, Gisbert Schneider1, Kaspar P. Locher2, Renato Zenobi1
- Department of Chemistry and Applied Biosciences, ETH Zürich, Vladimir-Prelog-Weg 3, 8093 Zürich, Switzerland
- Institute of Molecular Biology and Biophysics, ETH Zürich, Otto-Stern-Weg 5, 8093 Zürich, Switzerland
- VIB-VUB Center for Structural Biology and Structural Biology Brussels, Vrije Universiteit Brussels, Pleinlaan 2, 1050 Brussels, Belgium
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.
CovalX Technology Used (Click each option to learn more)
Samples of Nb80, Nb84, Nb87, Nb93, and Nb97 were subjected to a cross-linking reaction with 18 μM PglK and then spotted onto a stainless steel MALDI plate using the sandwich spotting technique with sinapic acid as the matrix. The analysis was performed on a MALDI-TOF/TOF mass spectrometer modified with the CovalX HM2 detection system. The use of high-mass MALDI-MS allowed researchers the ability to characterize intact Nb membrane protein complexes. Specifically, the stoichiometric distribution the complexes formed in solution was used, which gave insight into the definitive conditions of the solution where homogeneous complex states are present.