In Situ Continuous Wave Electron Paramagnetic Resonance Investigation of the Amyloid Aggregation of Parkinson’s Protein Alpha-Synuclein—the Second Spin-Label Position

Self-aggregation of amyloid proteins is a crucial step in neurodegenerative disease. The protein alpha-synuclein (αS) is implicated in Parkinson’s disease. In an extension of the demonstration of in situ observation of intermediates in αS-aggregation by continuous wave (cw) EPR at room temperature (Zurlo et al. PLoS One 16: e0245548, 2021) by spin-label EPR, here the spin label is attached to position 90 (R1αS90), rather than at position 56. The aim is to determine, if the spin-label position affects the kinetics of aggregation and if local information on the intermediates is accessible. Probed by the MTSL ((1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate) spin label at position 90, using diamagnetic dilution of 9:1 wild type αS to R1αS90, similar aggregation kinetics are found. Rotation correlation times for the spin label in the oligomer cannot be determined with sufficient accuracy to obtain local information on the oligomer under the conditions used. At the present stage, higher resolution EPR approaches, such as high-field EPR are more promising.

A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

A novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER Distance Restraints

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

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.