The contribution of modern EPR to structural biology

Abstract

Electron paramagnetic resonance (EPR) spectroscopy combined with site-directed spin labelling is applicable to biomolecules and their complexes irrespective of system size and in a broad range of environments. Neither short-range nor long-range order is required to obtain structural restraints on accessibility of sites to water or oxygen, on secondary structure, and on distances between sites. Many of the experiments characterize a static ensemble obtained by shock-freezing. Compared with characterizing the dynamic ensemble at ambient temperature, analysis is simplified and information loss due to overlapping timescales of measurement and system dynamics is avoided. The necessity for labelling leads to sparse restraint sets that require integration with data from other methodologies for building models. The double electron-electron resonance experiment provides distance distributions in the nanometre range that carry information not only on the mean conformation but also on the width of the native ensemble. The distribution widths are often inconsistent with Anfinsen's concept that a sequence encodes a single native conformation defined at atomic resolution under physiological conditions.

Keywords: EPR spectroscopy; crystallography; intrinsically disordered proteins; molecular modelling; protein structure; transmembrane proteins.

Figure 1.. Label-to-label distance distributions reveal backbone disorder.

(a) Distribution of the Cα–Cα backbone distance r_αα (blue) and the label-to-label distance r_ee (red) for residue pair 96/143 in the rigid core of plant light-harvesting complex LHCII. The width of P(r_ee) is well below 10 Å. (b) Distributions for residue pair 3/34 in the flexible N-terminal domain. Label-induced broadening is enhanced by the variation of side chain orientations of disordered residue 3. The width of P(r_ee) is well above 10 Å. The distributions were simulated in MMM [26] based on PDB structure 2BHW and the restraints reported in [53].

Figure 2.. Entropic tuning of affinity by coupling an order–disorder transition to binding.

(a) For a too low-affinity combination of ligand and binding site, coupling to an order–disorder transition increases the free energy gain upon binding. (b) For a too high-affinity combination, coupling to a disorder–order transition reduces the free energy gain.