Long-range distances in amyloid fibrils of α-synuclein from EPR spectroscopy

August 21, 2013

Understanding the molecular structure and interactions of biomolecules relies on the availability of suitable spectroscopic methods. Electron spin resonance (EPR) can deliver important distance information at the atomic and nanometer length scale not accessible by other techniques. One of the most important recent developments in EPR spectroscopy was the establishment of methods to measure distances in biomolecules up to 10 nm with a precision of a few angstroms. In diamagnetic proteins, the method requires the introduction of diluted paramagnetic spin probes that are possibly small and non-interfering with the protein structure. This is usually accomplished by small organic molecules, derivatives of nitroxide radicals, which are reactive to cysteine side-chains by formation of a disulfide bond. To measure long-range distances between two specific residues in proteins, cysteines are introduced pairwise by site-directed mutagenesis. Nevertheless, in complex bio-macromolecules spin labeling strategies have to be developed in order to take individual biophysical and biochemical properties of the target protein into account.

EPR experiments can measure the dipolar coupling between electron spins, which is related to their inter-spin distance (left). Spin labels are inserted pairwise in monomeric α-synuclein and the protein is diluted with wt (1:30) prior to in-vitro aggregation to amyloid fibrils.

Amyloid fibrils of α-synuclein are pathological hallmarks of Parkinson’s and other neurodegenerative diseases. Several recent studies point on the role of this protein’s oligomeric species as well as on the fibrils for the intercellular spread of the disease. Magnetic resonance methods are particularly valuable to gain insights into the structure of these molecular aggregates due to lack of information on the molecular level e.g. from X-ray structures. On one hand, long-range distance information from EPR spectroscopy can ideally complement short-range distances from nuclear magnetic resonance (NMR), thereby providing the basis of a high-resolution structure. On the other hand, the labeling procedure for EPR spectroscopy is disputable due to the intrinsic instability of these proteins with respect to point mutations.

EPR dipolar oscillation traces (right) and extracted inter-spin distances between pairwise labeled amino acid residues in α-synuclein fibrils. Distances between residues on the external strand and on inner strands are reported as a function of the amino acid sequence (left).

In our recent paper highlighted here, we have investigated the formation of α-synuclein fibrils after pairwise introduction of spin labels in the monomeric protein and controlled aggregation under suitable dilution conditions. This was monitored combining different techniques including ThioT fluorescence measurements and electron microscopy. For several samples we were able to obtain highly homogeneous fibrils that allowed EPR distance measurements between largely conserved β-strand regions. Taking advantage of the structural properties of β-strands, we gained vectorial information on the spatial arrangements of the labels within the strands. The results provided a considerable number of long-range constrains on the fold of α-synuclein in fibrils and demonstrated the capability of EPR spectroscopy to access information on these aggregates. Detection of inter-strand distances in fibrils will potentially allow for extending these measurements to oligomeric states of these protein families and obtaining important insight into their role in misfolding and diseases. (mb)

Go to Editor View