Assessing dimerisation degree and cooperativity in a biomimetic small-molecule model by pulsed EPR

Abstract

Pulsed electron paramagnetic resonance (EPR) spectroscopy is gaining increasing importance as a complementary biophysical technique in structural biology. Here, we describe the synthesis, optimisation, and EPR titration studies of a spin-labelled terpyridine Zn(II) complex serving as a small-molecule model system for tuneable dimerisation.

Quantitative Resolution of Monomer‐Dimer Populations by Inversion Modulated DEER EPR Spectroscopy

“…The modulation depth of a DEER echo curve (Figure 1 B) provides a means of spin counting [3] with applications to both organic radicals and biomolecules. [4] Previous work attempted quantification of protein dimerization based on one-point measurements of modulation depth for singly spin-labeled mutants, relying on calibration relative to bi- and tri-radicals. [5] Here we show that analysis of modulation depth as a function of the electron double resonance (ELDOR) pulse flip angle (Figure 1 A) can be used to accurately quantify monomer/dimer populations in an equilibrium mixture (frozen out at low temperature).…”

“…[4ab] For a homogeneous distribution of spin-labeled molecules in a glassy frozen solution, B ( t ) can be described by a single exponential decay [7] The difference between the values of V ( t=0 ) and B ( t =0) is the modulation depth Δ (Figure 1 B). …”

“…The ratio of modulation depth, Δ( θ ), at a given ELDOR pulse flip angle θ , to the maximum modulation depth, Δ max , obtained at θ = 180°, is given by Equation (1): [4b] $${\left[\mathrm{\Delta }(\theta )/{\mathrm{normal\Delta }}_{max}\right]}_N=\frac{1={\left[1-{\lambda }_{max}\left(1-\text{cos}\theta \right)/2\right]}^{N-1}}{1-{\left(1-{\lambda }_{max}\right)}^{N-1}}$$ where λ max is the inversion efficiency at θ =180°, and N the number of nitroxide spin labels. For a doubly nitroxide spin-labeled sample, N = 2 for a monomer, 4 for a dimer, 6 for a trimer, and so on.…”

“…The observed normalized modulation depth (Δ/Δ max ) obs is given by a population-weighted average of the different species present in the EPR sample. Thus for a mixture of monomer and dimer with 100% double nitroxide spin labeling, (Δ/Δ max ) obs is given by Equation (2): [4b] $${\left(\mathrm{\Delta }/{\mathrm{normal\Delta }}_{max}\right)}_{\text{obs}}=p_{\mathrm{m}}{\left(\mathrm{\Delta }/{\mathrm{normal\Delta }}_{max}\right)}_{N=2}+\left(1-p_{\mathrm{m}}\right){\left(\mathrm{\Delta }/{\mathrm{normal\Delta }}_{max}\right)}_{N=4}$$ where p m is the monomer population. The formulation presented in Equations (1) and (2), in contrast to one that only looks at absolute modulation depth, [3] does not require calibration with model compounds containing a known number of spins.…”

“…The formulation presented in Equations (1) and (2), in contrast to one that only looks at absolute modulation depth, [3] does not require calibration with model compounds containing a known number of spins. [4b] If spin-labeling is incomplete, both two- and three-spin dimeric species have to be taken into account, and Equation (2) needs to be expanded to include terms for these species, as well as the labeling efficiency (Figure S2), which can easily be ascertained by mass spectrometry.…”