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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.Graphical A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0035-z) contains supplementary material, which is available to authorized users.
Highlights
Decades of research on protein–protein interactions have revealed valuable information about the structure and function of many protein complexes
The ensemble of orientations obtained on the basis of Paramagnetic relaxation enhancement (PRE) is represented by the iron atoms of each cytochrome c peroxidase (CcP) molecule, shown as red spheres
Paramagnetic nuclear magnetic resonance spectroscopy (NMR) and the PRE effect have enabled the characterization of a weak selfassociation between CcP molecules and provided restraints for modelling the complex using protein docking
Summary
Decades of research on protein–protein interactions have revealed valuable information about the structure and function of many protein complexes. Transient complexes include ultra-weak interactions, defined as having a dissociation constant (KD) in the millimolar range, (Tang et al 2008a, b; Vaynberg and Qin 2006) that are known to drive self-assembly of higher order homogeneous architectures like crystals, viral capsids and amyloid fibrils (Fawzi et al 2007; Garcia-Ruiz 2003; Zlotnick 2005). They play an important role in an array of cellular processes including rapid assembly/disassembly, protein maturation, reversible cell adhesion and cell signalling (Vaynberg and Qin 2006). These conformations are lowly populated, transient and cannot be isolated, making them practically invisible to conventional structural biology techniques (Clore 2011)
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