In situ kinetic measurements of α-synuclein aggregation reveal large population of short-lived oligomers.

Knowledge of the mechanisms of assembly of amyloid proteins into aggregates is of central importance in building an understanding of neurodegenerative disease. Given that oligomeric intermediates formed during the aggregation reaction are believed to be the major toxic species, methods to track such intermediates are clearly needed. Here we present a method, electron paramagnetic resonance (EPR), by which the amount of intermediates can be measured over the course of the aggregation, directly in the reacting solution, without the need for separation.

A Two-Armed Probe for In-Cell DEER Measurements on Proteins*.

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances.

Tracking amyloid oligomerization with monomer resolution using a 13-amino acid peptide with a backbone-fixed spin label.

Amyloid oligomers are suspected as toxic agents in neurodegenerative disease, and are transient and often heterogeneous, making them difficult to detect. Here we show an approach to track the development of amyloid oligomers in situ by room temperature, continuous wave (cw) 9 and 95 GHz EPR. Three amyloid peptides with the 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC) spin label were synthesized by solid phase peptide synthesis: T0EZ (TKVKVLGDVIEVGG) with TOAC (T) at the N-terminus, T5EZ with TOAC in the middle (KVKVTGDVIEVG) and T12EZ with TOAC at the C-terminus (KVKVLGDVIEVTG).

A tailored multi-frequency EPR approach to accurately determine the magnetic resonance parameters of dynamic nuclear polarization agents: application to AMUPol.

To understand the dynamic nuclear polarization (DNP) enhancements of biradical polarizing agents, the magnetic resonance parameters need to be known. We describe a tailored EPR approach to accurately determine electron spin-spin coupling parameters using a combination of standard (9 GHz), high (95 GHz) and ultra-high (275 GHz) frequency EPR. Comparing liquid- and frozen-solution continuous-wave EPR spectra provides accurate anisotropic dipolar interaction D and isotropic exchange interaction J parameters of the DNP biradical AMUPol.