Proteins & Protein Interactions

Prof. dr. Marcellus Ubbink

Proteins have to form complexes with DNA, other proteins and small molecules. We investigate how protein complexes are formed and what they look like at the atomic level. We have shown that some complexes are dynamic and we try to make a ‘movie’ of their interactions. We also determine how small molecules travel through an enzyme to reach the active site. This requires 'breathing' of the protein structure. When you consider such dynamics as an inherent property of proteins, the structures come to life. This research is so fascinating because it studies biomolecules really at the atomic level. It brings together physics, chemistry and biology to elucidate the physical-chemical properties that dictate how proteins work.

Transient protein complexes

The biological function of a protein-protein complex determines its lifetime. Some complexes must exist for a long time and be very tight. Others exist only fleetingly and dissociate rapidly. The functional requirements are met by different types of biophysical properties. We study the process of protein complex formation of short-lived and dynamic complexes, such as plastocyanin-cytochrome f and cytochrome c and cytochrome c peroxidase. We have elucidated the nature of the encounter states of such complexes using paramagnetic NMR spectroscopy.

Protein-ligand interactions

Enzymes convert small compounds and thus are common targets for drug development. We develop methods to characterize protein-ligand complexes in solution. Also here paramagnetic NMR can provide the information necessary for determination of the binding site and orientation of a ligand on a protein surface or in a protein active site.

Figure: The interface of cytochrome P450cam and putidaredoxin seen in the crystal structure of the complex

Paramagnetic NMR probes

Paramagnetic NMR represents a powerful toolbox for the characterisation of protein complexes. However, paramagnetism requires the presence of unpaired electrons in a sample. We have developed caged lanthanide NMR probes (CLaNPs) that can be attached to protein surfaces to provide the unpaired electrons in a highly controlled way. These probes find application for many types of protein NMR studies.

Figure: Ubiquitin labelled with CLaNP-5 – a model


1. The structure of cytochrome P450cam and its redox partner putidaredoxin was solved by attaching CLaNP to both proteins and obtaining more than 400 distance restraints for docking of the two proteins. It yielded a high quality structure, which was later confirmed by crystallography. The NMR study also provided evidence for an encounter complex that is present for a small fraction of the time.

Figure: A model of the encounter complex of cytochrome f and plastocyanin

2. The photosynthetic redox complexes of cytochrome f with plastocyanin and cytochrome c6 were characterized using paramagnetic NMR and modelling and were demonstrated to be highly dynamic. In both cases the encounter complex comprised both charge-charge and hydrophobic interactions, which makes it different from many other encounter complexes. These findings led to a new model of complex formation.

3. CLaNPs with various chemical and paramagnetic properties have been developed. They all feature two arms for rigid attachment to a protein surface carrying two cysteine residues. As a consequence, superior paramagnetic data are obtained with large effects and highly predictable lanthanide positions.

Figure: CLaNP-5 on the surface of cytochrome c – a model

  1. Skinner, S.P., W.-M. Liu, Y. Hiruma, M. Timmer, A. Blok, M.A.S. Hass, M. Ubbink, "Delicate conformational balance of the redox enzyme cytochrome P450cam", Proceedings of the National Academy of Sciences, vol. 112, issue 29, pp. 9022 - 9027, 07/2015. DOI: 10.1073/pnas.1502351112
  2. Hiruma, Y., C. Sacristan, S.T. Pachis, A. Adamopoulos, T. Kuijt, M. Ubbink, E. von Castelmur, A. Perrakis, et al., "Competition between MPS1 and microtubules at kinetochores regulates spindle checkpoint signaling", Science, vol. 348, issue 6240, pp. 1264 - 1267, 06/2015. DOI: 10.1126/science.aaa4055
  3. Hiruma, Y., M.A.S. Hass, Y. Kikui, W.M. Liu, B. Olmez, S.P. Skinner, A. Blok, A. Kloosterman, et al., "The Structure of the Cytochrome P450cam-Putidaredoxin Complex Determined by Paramagnetic NMR Spectroscopy and Crystallography", Journal of Molecular Biology, vol. 425, no. 22, pp. 4353-4365, Nov 15, 2013. DOI: 10.1016/J.Jmb.2013.07.006
  4. Meschi, F., F. Wiertz, L. Klauss, A. Blok, B. Ludwig, A. Merli, H.A. Heering, G.L. Rossi, et al., "Efficient Electron Transfer in a Protein Network Lacking Specific Interactions", Journal of the American Chemical Society, vol. 133, issue 42, pp. 16861 - 16867, 10/2011. DOI: 10.1021/ja205043f
  5. Volkov, A.N., J.A.R. Worrall, E. Holtzmann, M. Ubbink, "Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR", Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 50, pp. 18945-18950, Dec 12, 2006. DOI: 10.1073/Pnas.0603551103

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