Abstract
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.
Highlights
The observed nuclear relaxation rates can either be used directly or after conversion into distances for structural modelling.[4,5]To convert the observed relaxation rates into distances, the correlation time of the vector r that connects the paramagnetic centre and the nucleus is required. tc depends of the rotational correlation time of this vector as well as the longitudinal electronic relaxation time, according to tcÀ1 = trÀ1 + tsÀ1.4,5 For spin labels, the contribution of ts is small and tc is dominated by tr
In order to compare the mobility of methane thiosulfonate (MTSL) and pyMTSL, these spin labels (SLs) were attached to three positions on the surface of cytochrome c peroxidase (CcP), positions 38, 200 and 288 (Fig. 2)
The electron paramagnetic resonance (EPR) spectra of the spin labels at the three positions investigated show that the lineshapes of the nitroxides are under conditions of not fully averaged anisotropy, typical for spin-labels attached to proteins
Summary
The differences between the free energies of SL conformations are often smaller than the thermal energy in the sample, meaning that the position of the unpaired electron is spatially distributed over an area determined by the occupied SL conformer orientations.[6] Over- or underestimation of the spatial distribution of the free electron will lead to errors in the apparent mean distance, [hrÀ6iÀ1/6].7. To solve this problem, the SL can be treated as an Over- or underestimation of the spatial distribution of the free electron will lead to errors in the apparent mean distance, [hrÀ6iÀ1/6].7 To solve this problem, the SL can be treated as an
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