Protein docking using an ensemble of spin labels optimized by intra-molecular paramagnetic relaxation enhancement

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.

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

“…Yeast [ 15 N, 2 H]- or [ 15 N, 13 C]-labelled CcP C128A with MSKT as the first four N-terminal residues was expressed and purified as published previously (Morar et al 1999 ; Pollock et al 1998 ; Schilder et al 2014 ). The same CcP construct with the additional mutations N38C, N200C or T288C were used to produce unlabelled protein (Schilder et al 2015 ; Volkov et al 2006 ). 1-acetoxy-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl)-methanethiosulfonate (MTS) and 1-oxyl-2,2,5,5-tetramethyl-2,5-dihydropyrrol-3-ylmethyl methanethiosulfonate (MTSL) tags were obtained from Toronto Research Chemicals (Toronto, ON, Canada).…”

New NMR tools for protein structure and function: Spin tags for dynamic nuclear polarization solid state NMR

“…This cysteine can be native to the protein, or introduced via mutagenesis, enabling most positions in the protein to be labeled. MTSL was the first probe used for in vitro structural determination of a protein using paramagnetic NMR restraints [98], and MTSL tags with tailored properties, such as greater rigidity, have been synthesized [99]. Other cysteine attachment chemistries, such as maleimide [100], have been explored as well.…”

NMR Signal Quenching from Bound Biradical Affinity Reagents in DNP Samples

“…Moreover, the distances calculated from R 2PRE are fairly insensitive to the motional effects, permitting an approach that only considers the ensemble effect of the motions to be acceptable for some applications. 65,68,70 …”