PELDOR in rotationally symmetric homo-oligomers

Abstract

Nanometre distance measurements by pulsed electron–electron double resonance (PELDOR) spectroscopy have become an increasingly important tool in structural biology. The theoretical underpinning of the experiment is well defined for systems containing two nitroxide spin-labels (spin pairs); however, recently experiments have been reported on homo-oligomeric membrane proteins consisting of up to eight spin-labelled monomers. We have explored the theory behind these systems by examining model systems based on multiple spins arranged in rotationally symmetric polygons. The results demonstrate that with a rising number of spins within the test molecule, increasingly strong distortions appear in distance distributions obtained from an analysis based on the simple spin pair approach. These distortions are significant over a range of system sizes and remain so even when random errors are introduced into the symmetry of the model. We present an alternative approach to the extraction of distances on such systems based on a minimisation that properly treats multi-spin correlations. We demonstrate the utility of this approach on a spin-labelled mutant of the heptameric Mechanosensitive Channel of Small Conductance of E. coli.

Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR

“…Nevertheless, the same trend could be reproduced using simulations based on geometric models mimicking 1 as a tetrahedron and MscS S196R1 as a regular convex heptagon15 (see Figure 3 and the Supporting Information). This proves that ‘Ghost Peaks’ and improper amplitudes in the distance distributions obtained with increasing λ arise from multi‐spin effects, as the observation time in the simulations was chosen long enough to avoid truncation artifacts.…”

“…This has been experimentally demonstrated for systems with up to three spins. However, performance of this approach reduces with increasing λ and n 7, 15. For more than four spins a combination of reducing λ and power‐scaling has been recommended 16.…”

“…These findings could also be confirmed on a newly synthesized rectangular tetraradical and an octameric Wza14 (see the Supporting Information). Furthermore, by systematically exploring the recovery of distance distributions from simulated data for equilateral triangles to octagons shows that power‐scaling can reliably recover the ‘true’ distance distribution as long as λ is kept below the maximum of the two‐spin contribution (1/( n ‐1)) 15, 16. Noise‐free simulations suggest that even larger λ might be tolerated but this is not confirmed upon addition of 1 to 3 % noise to the simulations (see the Supporting Information).…”

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

“…This is a typical manifestation of multispin effects in DEER results. Multispin interactions have been shown to be more disruptive in high-order oligomers than in lower-order oligomers (Giannoulis et al, 2013), which explains why the effect is so strong in these hexameric assemblies. Nevertheless, the low SNR and short dipolar evolution times can also contribute to these effects.…”

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

“…The DeerAnalysis and MMM simulation programs developed by Jeschke et al [39] are excellent tools used by many scientists to quantitatively analyze and interpret such experimental data and to compare the results with predictions based on X-ray structures or models. If more than two spin labels are attached to the macromolecule, the interpretation of the PELDOR time traces become more cumbersome [40,41]. However, valuable information on the number of spin labels, for example the oligomeric state of protein complexes can still be obtained [42,43].…”