The architecture of multi-domain proteins is crucial for their function. However, the determination of such structures in native-like environments to high resolution remains difficult. Electron Paramagnetic Resonance (EPR) spectroscopy is a highly sensitive technique that can be used to study samples in near native-like conditions using small sample volumes. Proteins are site-specifically labelled with nitroxides and Pulsed Electron-electron Double Resonance (PELDOR) is used to measure the interactions between pairs of nitroxide spin labels. These interactions can be interpreted as a distribution of distances (in the range 2 to 8 nm) between the two nitroxide labels bound to the protein, which includes information on the structure and dynamics of both the biomolecule and the nitroxide label. It is possible to utilize these distance distributions as distance constraints to assist computational structure refinement in order to deduce the architecture of multi-domain proteins.
The cover image shows the crystal structure of the three POTRA domains of anaOmp85 from the cyanobacterium Anabaena sp. PCC 7120 (PDB code: 3MC8). The nitroxide spin-labels used in our study have been added to the original crystal structure, each site is represented by a different coloured collection of rotamers. These were derived from molecular dynamics simulations performed with YASARA and depict the intrinsic flexibility of the label used. An experimental data set, so-called PELDOR time trace, which oscillates with a frequency dependent on the interaction and distance between the nitroxide labels, is shown in the lower left corner. The long lasting and pronounced modulations of that signal are remarkable for a biological system. Distance distributions are extracted from the time domain signals using a mathematical approach called Tikhonov regularization. The distribution corresponding to the trace shown is displayed in the upper right corner.
In our study (Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy. Biophysical Journal 110/10) we describe the application of these distance distributions as distance constraints in structure refinement unravelling a restricted conformational flexibility of the POTRA domains compared to unrestricted molecular dynamics simulations. The structures we derive also show a change in the architecture of the domains compared to the crystal structure, which may be a result of the conditions under which crystallization was achieved.
The combination of distance restraints from PELDOR with structural refinement has the potential to be applied in many different protein systems and therefore is a valuable tool in the armoury of protein structure refinement techniques.
– Reza Dastvan, Eva-Maria Brouwer, Denise Schuetz, Oliver Mirus, Enrico Schleiff, Thomas Prisner