Protein engineering and structural biology
To understand how proteins function, we need to know their three-dimensional structure. We use two techniques to determine such 3D structures of proteins and their complexes: X-ray crystallography and cryogenic electron microscopy (cryo-EM).
For X-ray crystallography, pure proteins or protein complexes are crystallized, which allows performing an X-ray diffraction experiment. The structure can then be calculated from the diffraction pattern.
For cryo-EM, protein is rapidly cooled so that the solution becomes vitreous. The 3D structure is determined by recording large imaging datasets of such films in the electron microscope under cryogenic conditions and using these to reconstruct a 3D contour (density) map.
Furthermore, we use different protein engineering techniques, both classical as well as recent AI-based (e.g. de novo design) methods, to stabilize transient and dynamic complexes, that would otherwise resist structure determination. We apply these techniques to study important blood protein complexes and understand their molecular mechanisms.
Research lines
In our lab, we have several research lines related to protein engineering, structure and function.
First of all, we are working on developing new tools for protein purification. We recently determined the amino acid sequence of the widely used (in >120,000 publications) anti-FLAG M2 antibody using mass spectrometry, which allowed us to rebuild an incomplete crystal structure of this antibody (Peng, Pronker and Snijder, JPR, 2021). We also generated plasmids to recombinantly produce anti-FLAG M2 based on this sequence (available through Addgene, here and here) and compared binding of such recombinantly produced anti-FLAG M2 with a commercially available alternative.
Subsequently, we crystallized the Fab of our recombinantly produced anti-FLAG M2 in complex with a FLAG peptide to determine the molecular determinants of recognition for this widely used tool. These crystals diffracted to very high (atomic) resolution (1.17 Å), allowing us to determine the structure of the complex, which also happened to be the highest-resolution structure of a Fab-antigen complex to date (Beugelink et al., JMB, 2024). The structure unexpectedly revealed that the FLAG peptide binds to anti-FLAG M2 in a 310 helix conformation. Furthermore, the experimental structure showed that the two C-terminal residues of the FLAG peptide did not (directly) contribute to the binding to anti-FLAG M2 as they were not resolved in the electron density. We measured the binding affinity of various variants of both anti-FLAG M2 and the FLAG peptide using surface plasmon resonance, which revealed that a shorter variant of the FLAG tag could bind with the same affinity to anti-FLAG M2 as the original. Currently, we are using computational protein design methods to explore alternative protein purification tag systems that do not rely on expensive mammalian cell culture to produce the antibody or synthetic peptides for mild elution.
We are also setting up computational pipelines for de novo design of minibinders and single domain antibodies (sdAbs, also known as VHHs), using the latest in silico methods. We will use these to purify (e.g. from endogenous material) and stabilize transient and dynamic complexes that resist structure determination by standard techniques such as X-ray crystallography. Next, we will determine the 3D structure of such stabilized complexes involved in hemostasis and immunity by cryo-EM, which will help us understand how they (mis)function. This will allow the development of next-generation therapeutics for various potentially deadly conditions such as myocardial infarction, thrombosis and bleeding disorders.
Key publications
- Beugelink, J.W., Sweep, E., Janssen, B.J.C., Snijder, J.** and Pronker, M.F.** (2024) Structural basis for recognition of the FLAG-tag by anti-FLAG M2 Journal of Molecular Biology, https://doi.org/10.1016/j.jmb.2024.168649
- Pronker, M.F., Creutznacher, R., Drulyte, I., Hulswit, R.J.G., Li, Z., van Kuppeveld, F.J.M., Snijder, J., Lang, Y., Bosch, B.J., Boons, G.J., Frank, M., de Groot, R.J., and Hurdiss, D.L. (2023) Sialoglycan binding triggers spike opening in a human coronavirus Nature, https://doi.org/10.1038/s41586-023-06599-z
- Peng, W.*, Pronker, M.F.*, and Snijder, J. (2021) Mass spectrometry-based de novo sequencing of monoclonal antibodies using multiple proteases and a dual fragmentation scheme Journal of Proteome Research, https://doi.org/10.1021/acs.jproteome.1c00169
- Pronker, M.F.**, van den Hoek, H., and Janssen, B.J.C.** (2019) Design and structural characterisation of olfactomedin-1 variants as tools for functional studies BMC Molecular and Cell Biology, https://doi.org/10.1186/s12860-019-0232-1
- Pronker, M.F., Lemstra, S., Snijder, J., Heck, A.J.R., Thies-weesie, D.M.E., Pasterkamp, R. J., and Janssen, B. J. C. (2016) Structural basis of myelin-associated glycoprotein adhesion and signalling Nature Communications, https://doi.org/10.1038/ncomms13584
- Pronker, M.F., Bos, T.G.A.A., Sharp, T.H., Thies-Weesie, D.M.E., and Janssen, B.J.C. (2015) Olfactomedin-1 has a V-shaped disulfide-linked tetrameric structure Journal of Biological Chemistry, https://doi.org/10.1074/jbc.m115.653485
* equal contribution , ** co-corresponding authors
Funding
Joghem van Loghem fellowship (2026-2030)
Ancillary positions
- Member of Dutch Society on Thrombosis and Haemostasis (NVTH)
- Member of Netherlands Society for Biochemistry and Molecular Biology (NVBMB)
- Member of the Dutch Crystallographic Society (NVK)
- Member of the Royal Netherlands Chemistry Society (KNCV)
