Oligomerization of integral membrane proteins is a common biophysical phenomenon that is often the final step of protein folding into functional assemblies. Stability of protein oligomers varies. While some oligomeric assemblies could persist under a broad range of experimental conditions including solubilization in detergents, it is not uncommon for the oligomeric structures to be affected by the membrane lipid composition and even sample preparation procedures. These considerations warrant further development of experimental methods suitable for quantitation and structure determination of protein oligomers in the native lipid milieu.
In a collaboration with laboratories of Profs. Brown and Ladizhansky (Department of Physics and Biophysics Interdepartmental Group, University of Guelph, Guelph, Ontario, CANADA) and Prof. Wang (Peking University, Beijing National Laboratory for Molecular Sciences (BNLMS), Beijing, People’s Republic of China) we are developing methods to combine spin-labeling Double Electron-Electron Resonance (DEER) at Q-band (34 GHz) and solid-state NMR (ssNMR) spectroscopy to refine the structure of a membrane-embedded oligomer proteins such as a trimer (~81 kDa) formed by seven-helical transmembrane photoreceptor Anabaena Sensory Rhodopsin (ASR, ~27 kDa) from Anabaena sp. PCC7120. ASR is a notable example of a membrane protein whose oligomeric state depends on the membrane mimetic environment. While ASR monomers have been shown to form stable trimers in cellular E.coli membranes1 and preserving the trimeric structure upon subsequent solubilization in detergents and upon reconstitution in lipids,2 the crystallization conditions appear to promote formation of dimers observed in the ASR crystals.3 An essential feature of a combined DEER + ssNMR approach employed here is that it provides structural distance restraints spanning a range of ca. 3-60 Å, while using the same sample preparation, such as site-directed mutations, paramagnetic labeling, and reconstitution in lipid bilayers, for both ssNMR and DEER. We also developed methods for direct modelling of the multispin effects on the DEER signal to determine the oligomeric order and obtain long-range DEER distance restraints between the ASR trimer subunits that were used to refine solid-state NMR structure of ASR.4 The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface when compared to the structure determined using solely the ssNMR data. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. We belive that our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization as well as to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.
- Ward, M. E.; Wang, S.; Munro, R.; Ritz, E.; Hung, I.; Gor’kov, P. L.; Jiang, Y.; Liang, H.; Brown, L. S.; Ladizhansky, V. Biophysical Journal 2015, 108, 1683.
- Wang, S.; Munro, R. A.; Shi, L.; Kawamura, I.; Okitsu, T.; Wada, A.; Kim, S. Y.; Jung, K. H.; Brown, L. S.; Ladizhansky, V. Nature Methods 2013, 10, 1007.
- Vogeley, L.; Sineshchekov, O. A.; Trivedi, V. D.; Sasaki, J.; Spudich, J. L.; Luecke, H. Science 2004, 306, 1390.
- Milikisiyants, S.; Wang, S.; Munro, R. A.; Donohue, M.; Ward, M. E.; Bolton, D.; Brown, L. S.; Smirnova, T. I.; Ladizhansky, V.; Smirnov, A. I. Journal of molecular biology 2017, 429, 1903.