Density functional theory studies of MTSL nitroxide side chain conformations attached to an activation loop

A quantum-mechanical (QM) method rooted on density functional theory (DFT) linked to the Stochastic Liouville equation (SLE) in the Fokker Planck (FP) form has been employed for the first time to sample the methane-thiosulfonate spin label (MTSL) conformational space attached to the Aurora-A kinase activation loop and to calculate the EPR spectrum. The features of the calculated energy surface allowed us to describe the system in a limited number of rotamers stabilized by interactions of the MTSL side chain and neighbouring residues. The relevant magnetic parameters and the electron paramagnetic resonance (EPR) spectrum were subsequently calculated from the trajectories of the spin probe in the protein environment. The comparison between theoretical and experimental continuous wave (CW) EPR spectra revealed some small differences in the EPR line shape which arises from the combinations of g- and A-values obtained from the conformations selected. The theoretical approach adopted in this work can be used to recognise the contribution of MTSL rotamers to the EPR spectrum in order to help extract structural/dynamics properties of protein from the experimental data

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies