Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography

Edward A. FitzGerald; Daniela Cederfelt; Daria Kovryzhenko; Pierre Boronat; Bjarte Aarmo Lund; Doreen Dobritzsch; Sven Hennig; Pablo Porragas Paseiro; Iwan J.P. De Esch; U. Helena Danielson

doi:10.1039/d5cb00041f

Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography

Edward A. FitzGerald
, Daniela Cederfelt
, Daria Kovryzhenko
, Pierre Boronat
, Bjarte Aarmo Lund
, Doreen Dobritzsch
, Sven Hennig
, Pablo Porragas Paseiro
, Iwan J.P. De Esch
, U. Helena Danielson^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

Original language	English
Pages (from-to)	1625-1639
Number of pages	15
Journal	RSC Chemical Biology
Volume	6
Issue number	10
Early online date	13 Aug 2025
DOIs	https://doi.org/10.1039/d5cb00041f
Publication status	Published - 1 Oct 2025

Bibliographical note

Publisher Copyright:
© 2025 RSC.

Funding

We wish to acknowledge Prof. Chris Ulens, Laboratory of Structural Neurobiology, KU Leuven for initial protein samplesand AChBP expression plasmids, Vladimir O. Talibov for help in initiating crystallization trials, Margaret Butko and Biodesy (Inc., South San Francisco, CA, United States) for providing access to SHG biosensor and assistance in performing experiments, and Matthias Molnar and NanoTemper Technologies (GmbH., Munich, Germany) for providing access to the Seismos SAW biosensor and advice for designing and interpreting experiments. The authors would like to acknowledge Dr Wouter Engelen at Dynamic Biosensors for his contributions to the DNA nanolever design and for technical advice throughout the switchSENSE measurements. Access to Diamond Light Source(Didcot, Oxfordshire, UK) was provided via the Dutch MX BAG.We thank the user support team as well as the beamline staff of beamline I04 for their support. We acknowledge the MAX IVLaboratory for beamtime on the Bio MAX beamline under proposal 20190180. Research conducted at MAX IV, a Swedishnational user facility, is supported by Vetenskapsrådet (Swedish Research Council, VR) under contract 2018-07152, Vinnova (Swedish Governmental Agency for Innovation Systems) under contract 2018-04969 and Formas under contract 2019-02496. This project has received funding from the European Union’s Framework Programme for Research and Innovation Horizon 2020 (2014–2020) under the Marie-Skoldowska-Curie Action. Edward Fitzgerald was part of FRAGNET, supported by grant agreement number ID 675899. Pablo Porragas Paseiro is part of SYNSENSO, supported by grant agreement number ID 101072980, and the UK Engineering and Physical Sciences Research Council. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research ExecutiveAgency. Neither the European Union nor the granting authority can be held responsible for them. B. A. L. thanks The Research Council of Norway for the Centre of Excellence and project grants (grant no. 262695 and 274858). Science for Life Laboratory, SciLifeLab Technology Development Project (TDP) 2019–2020 We wish to acknowledge Prof. Chris Ulens, Laboratory of Structural Neurobiology, KU Leuven for initial protein samples and AChBP expression plasmids, Vladimir O. Talibov for help in initiating crystallization trials, Margaret Butko and Biodesy (Inc., South San Francisco, CA, United States) for providing access to SHG biosensor and assistance in performing experiments, and Matthias Molnar and NanoTemper Technologies (GmbH., Munich, Germany) for providing access to the Seismos SAW biosensor and advice for designing and interpreting experiments. The authors would like to acknowledge Dr Wouter Engelen at Dynamic Biosensors for his contributions to the DNA nanolever design and for technical advice throughout the switchSENSE measurements. Access to Diamond Light Source (Didcot, Oxfordshire, UK) was provided via the Dutch MX BAG. We thank the user support team as well as the beamline staff of beamline I04 for their support. We acknowledge the MAX IV Laboratory for beamtime on the BioMAX beamline under proposal 20190180. Research conducted at MAX IV, a Swedish national user facility, is supported by Vetenskapsrådet (Swedish Research Council, VR) under contract 2018-07152, Vinnova (Swedish Governmental Agency for Innovation Systems) under contract 2018-04969 and Formas under contract 2019-02496. This project has received funding from the European Union's Framework Programme for Research and Innovation Horizon 2020 (2014–2020) under the Marie-Skoldowska-Curie Action. Edward Fitzgerald was part of FRAGNET, supported by grant agreement number ID 675899. Pablo Porragas Paseiro is part of SYNSENSO, supported by grant agreement number ID 101072980, and the UK Engineering and Physical Sciences Research Council. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. B. A. L. thanks The Research Council of Norway for the Centre of Excellence and project grants (grant no. 262695 and 274858). Science for Life Laboratory, SciLifeLab Technology Development Project (TDP) 2019–2020.

Funders	Funder number
KU Leuven
Horizon 2020 Framework Programme
Engineering and Physical Sciences Research Council
Vetenskapsrådet	2018-07152
NanoTemper Technologies	20190180
VINNOVA	2018-04969
Svenska Forskningsrådet Formas	2019-02496
H2020 Marie Skłodowska-Curie Actions	675899, ID 101072980
Norges forskningsråd	262695, 274858

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FitzGerald, E. A., Cederfelt, D., Kovryzhenko, D., Boronat, P., Lund, B. A., Dobritzsch, D., Hennig, S., Paseiro, P. P., De Esch, I. J. P., & Danielson, U. H. (2025). Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography. RSC Chemical Biology, 6(10), 1625-1639. https://doi.org/10.1039/d5cb00041f

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title = "Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography",

abstract = "Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.",

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FitzGerald, EA, Cederfelt, D, Kovryzhenko, D, Boronat, P, Lund, BA, Dobritzsch, D, Hennig, S, Paseiro, PP, De Esch, IJP & Danielson, UH 2025, 'Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography', RSC Chemical Biology, vol. 6, no. 10, pp. 1625-1639. https://doi.org/10.1039/d5cb00041f

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AU - FitzGerald, Edward A.

AU - Cederfelt, Daniela

AU - Kovryzhenko, Daria

AU - Boronat, Pierre

AU - Lund, Bjarte Aarmo

AU - Dobritzsch, Doreen

AU - Hennig, Sven

AU - Paseiro, Pablo Porragas

AU - De Esch, Iwan J.P.

AU - Danielson, U. Helena

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N2 - Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

AB - Analysis of ligand-induced structural changes in proteins is challenging due to the lack of experimental methods suited for detection and characterisation of both ligand binding and induced structural changes. We have explored biosensors with different detection principles to study interactions between ligands and acetylcholine binding proteins (AChBPs), soluble homologues of Cys-loop ligand gated ion channels (LGICs) that undergo similar structural changes as LGICs upon ligand binding. X-ray crystallography was used to identify binding sites and establish if the detected conformational changes involved small changes in loop C or major structural changes in the pentamer associated with ion channel opening. Experiments were initially focused on ligands exhibiting complex surface plasmon resonance (SPR) biosensor sensorgrams or detected by second harmonic generation (SHG) biosensor analysis. Surface acoustic wave (SAW) and SHG biosensors confirmed that complexities in SPR data were indeed due to ligand-induced conformational changes. Grating coupled interferometry (GCI) biosensor sensorgrams were less complex, despite similar detection principles. switchSENSE biosensor analysis revealed that ligands resulted in either a compaction or expansion of the protein structure. X-ray crystallography of the protein–ligand complexes was only successful for 7 out of 12 ligands, despite nM–μM affinities. Crystals were not obtained for the two compounds shown by SHG analysis to induce large structural changes, while electron densities were not seen in the structures for some ligands. The work presented herein shows that several biosensor technologies have a unique capability to detect and discriminate binding and ligand induced conformational changes in proteins, also when interactions are rapid, weak and structural changes are small. However, they are complementary and provide different information.

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JF - RSC Chemical Biology

IS - 10

ER -

Detection and characterisation of ligand-induced conformational changes in acetylcholine binding proteins using biosensors and X-ray crystallography

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