Weak self-association of cytochrome c peroxidase molecules observed by paramagnetic NMR

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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 1â??25Â*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 CcPâ??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 â??backâ?? of CcP. The CcPâ??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 CcPâ??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 Abstract

A weak self-association between cytochrome c peroxidase molecules was characterized using paramagnetic NMR.



Source: Journal of Biomolecular NMR