Unveiling the invisible conformational landscape of cytochrome P450
- 2009 - 2014
- Marcellus Ubbink
- NWO Vici
Structural biology has greatly contributed to our understanding of molecular interactions. However, the structures always represent the low-energy, most abundant states of the molecules, while often high-energy states play a critical role in the biochemical process. The aim of the research is to visualize such lowly populated, "invisible" states. Cytochromes P450 (CYPs) represent an excellent example. This ubiquitous superfamily of heme enzymes is involved in oxidation of many endogenous substrates and most drugs. The active site is completely buried and structures of many CYPs show no access channels. Yet, somehow the substrate has to enter the active site. In solution, the closed state must be in dynamic exchange with a lowly populated, open state. The structure of such a minor state cannot be determined by X-ray diffraction or classical NMR spectroscopy. Marcellus Ubbink proposes to determine it by a new, advanced NMR approach, in which the researchers will combine relaxation dispersion NMR with paramagnetic probes that he has developed. This method offers great promise for the characterization of minor states of proteins in general.
Also the entry of electrons into the active site involves transient structures. CYP associates with partner proteins that donate the required electrons. Remarkably, the enzyme can vary the specificity of this interaction during the catalytic cycle. To visualize the underlying conformational changes, the structures of complexes of a CYP and its partner will be determined with probes that create long-range, intermolecular effects. I have pioneered this approach for structure determination of large complexes. Now, for the first time structural rearrangements upon binding will be resolved.
This research will enhance our understanding of catalytic control of a very important class of enzymes. The developed methods will enable structure determination of hitherto invisible forms of proteins and complexes, which can find wide application in other fields of protein chemistry.