title: Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR.
abstract: There is growing experimental evidence that many proteins exhibit a tendency for (ultra)weak homo- or hetero- oligomerization interactions. With the development of paramagnetic relaxation enhancement NMR spectroscopy it has become possible to characterize weak complexes experimentally and even detect complexes with affinities in the 1-25 mM range. We present evidence for a weak complex between cytochrome c peroxidase (CcP) molecules. In a previous study, we attached nitroxide based spin labels at three positions on CcP with the intent of observing intramolecular PRE effects. However, several intermolecular PRE effects were also observed suggesting a weak self-association between CcP molecules. The CcP-CcP complex was characterized using paramagnetic NMR and protein docking. The interaction occurs between the surface that is also part of the stereo-specific binding site for its physiological partner, cytochrome c (Cc), and several small, positively charged patches on the "back" of CcP. The CcP-CcP complex is not a stereo-specific complex. It is a dynamic ensemble of orientations, characteristic of an encounter state. The contact areas resemble those observed for CcP molecules in crystals. The CcP-CcP complex formation competes with that of the CcP-Cc complex. However, the affinity for Cc is much larger and thus it is expected that, under physiological conditions, auto-inhibition will be limited. A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands.
authors.forename: Jesika
authors.initials: J
authors.lastname: Schilder
authors.affiliation: Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, 2333 CC, Leiden, The Netherlands. m.ubbink@chem.leidenuniv.nl.
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D003578:Cytochrome-c Peroxidase
keywords: Cytochrome c; Electron transfer; Ensemble docking; Paramagnetic relaxation enhancement; Ultra-weak interactions