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.

Light-Induced Porphyrin-Based Spectroscopic Ruler for Nanometer Distance Measurements.

We present a novel pulsed electron paramagnetic resonance (EPR) spectroscopic ruler to test the performance of a recently developed spin-labeling method based on the photoexcited triplet state (S=1). Four-pulse electron double resonance (PELDOR) experiments are carried out on a series of helical peptides, labeled at the N-terminal end with the porphyrin moiety, which can be excited to the triplet state, and with the nitroxide at various sequence positions, spanning distances in the range 1.8-8 nm.

Linker dependent chirality of solvent induced self-assembled structures of porphyrin-α-helical peptide conjugates.

The solvent-promoted aggregation of porphyrins covalently linked to medium length peptides occurs with the formation of chiral supramolecular structures if the peptide chain can adopt an α-helical secondary structure. The circular dichroism spectra of different porphyrin-peptide conjugates show that the chiral arrangement of the porphyrins in the aggregates does not depend on the screw-sense of the peptide helix. Experimental evidence and molecular dynamic simulations suggest that the linker between the porphyrin and the peptide helix is responsible for the overall chirality of supramolecular structures.

Porphyrin triplet state as a potential spin label for nanometer distance measurements by PELDOR spectroscopy.

This work demonstrates, for the first time, the feasibility of applying pulsed electron-electron double resonance (PELDOR/DEER) to determine the interspin distance between a photoexcited porphyrin triplet state (S = 1) and a nitroxide spin label chemically incorporated into a small helical peptide. The PELDOR trace shows deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair, providing an accurate distance measurement. This new labeling approach has a high potential for measuring nanometer distances in more complex biological systems due to the sensitivity acquired from the spin polarization of the photoexcited triplet state spectrum.