a b s t r a c tFormation of an encounter complex is important for efficient protein complex formation. The encounter state consists of an ensemble of orientations of two proteins in the complex. Experimental description of such ensembles inherently suffers from insufficient data availability. We have measured paramagnetic relaxation enhancements (PRE) on cytochrome c peroxidase (CcP) caused by its partner cytochrome c (Cc) carrying a spin label. The data complement earlier PRE data of spin labelled CcP, identifying several new interactions. This work demonstrates the need of obtaining as many independent data sets as possible to achieve the most accurate description of an encounter complex.
Structured summary of protein interactions:CcP and Cc bind by nuclear magnetic resonance (View interaction)
Paramagnetic NMR is a useful technique to study proteins and protein complexes and the use of paramagnetic relaxation enhancement (PRE) for this purpose has become wide-spread. PREs are commonly generated using paramagnetic spin labels (SLs) that contain an unpaired electron in the form of a nitroxide radical, with 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydropyrrol-3-ylmethyl methane thiosulfonate (MTSL) being the most popular tag. The inherent flexibility of the SL causes sampling of several conformations in solution, which can be problematic as over- or underestimation of the spatial distribution of the unpaired electron in structural calculations will lead to errors in the distance restraints. We investigated the effect of this mobility on the accuracy of protein-protein docking calculations using intermolecular PRE data by comparing MTSL and the less mobile 3-methanesulfonilthiomethyl-4-(pyridin-3-yl)-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-1-yloxyl (pyMTSL) on the dynamic complex of cytochrome c and cytochrome c peroxidase. No significant differences were found between the two SLs. Docking was performed using either single or multiple conformers and either fixed or flexible SLs. It was found that mobility of the SLs is the limiting factor for obtaining accurate solutions. Optimization of SL conformer orientations using intra-molecular PRE improves the accuracy of docking.
NMR relaxation dispersion techniques provide a powerful method to study protein dynamics by characterizing lowly populated conformations that are in dynamic exchange with the major state. Paramagnetic NMR is a versatile tool for investigating the structures and dynamics of proteins. These two techniques were combined here to measure accurate and precise pseudocontact shifts of a lowly populated conformation. This method delivers valuable long-range structural restraints for higher energy conformations of macromolecules in solution. Another advantage of combining pseudocontact shifts with relaxation dispersion is the increase in the amplitude of dispersion profiles. Lowly populated states are often involved in functional processes, such as enzyme catalysis, signaling, and protein/protein interactions. The presented results also unveil a critical problem with the lanthanide tag used to generate paramagnetic relaxation dispersion effects in proteins, namely that the motions of the tag can interfere severely with the observation of protein dynamics. The two-point attached CLaNP-5 lanthanide tag was linked to adenylate kinase. From the paramagnetic relaxation dispersion only motion of the tag is observed. The data can be described accurately by a two-state model in which the protein-attached tag undergoes a 23° tilting motion on a timescale of milliseconds. The work demonstrates the large potential of paramagnetic relaxation dispersion and the challenge to improve current tags to minimize relaxation dispersion from tag movements.
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 AbstractA 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.
We present a novel approach to study transient protein-protein complexes with standard, 9 GHz, and high-field, 95 GHz, electron paramagnetic resonance (EPR) and paramagnetic NMR at ambient temperatures and in solution. We apply it to the complex of yeast mitochondrial iso-1-cytochrome c (Cc) with cytochrome c peroxidase (CcP) with the spin label [1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl)-methanethiosulfo-nate] attached at position 81 of Cc (SLÀ Cc). A dissociation constant K D of 20 � 4 × 10 À 6 M (EPR and NMR) and an equal amount of stereo-specific and encounter complex (NMR) are found. The EPR spectrum of the fully bound complex reveals that the encounter complex has a significant population (60 %) that shares important features, such as the Cc-interaction surface, with the stereo-specific complex.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.