Protein folding, membrane tubulation and nanoparticle-based solubilization

Michael Overduin3, Marc Lenoir3, Mohammed Jamshad1, Yu pin Lin1,Timothy Knowles3, Ali Jazayeri4, David Poyner2 , Roslyn Bill2, Mark Wheatley1, Timothy Dafforn1, Xinwang Cao5, Ünal Coskun5, Manfred Rössle6, Sabine B. Buschhorn5, Michal Grzybek5, Kai Simons5

School of Biosciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

School of Life and Health Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET,U.K.

School of Cancer Sciences, The University of Birmingham, Edgbaston, Birmingham, B15 2TT, U.K.

Heptares, BioPark^?, Broadwater Road, Welwyn Garden City, Hertfordshire, AL7 3AX, UK

Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstraße 108 Dresden, Germany

European Molecular Biology Laboratory EMBL, Hamburg Outstation c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany

A new nanoparticle method for stabilizing functionally intact membrane proteins has been optimized for solubilization of GPCRs directly from eukaryotic membranes. Using an amphipathic co-polymer composed of styrene maleic acid (SMA), the membrane was converted into soluble particles which contain native lipids and active membrane proteins, enabling analysis of their structural properties, oligomeric state and molecular functions. We have shown that this polymer can extract and preserve transmembrane phospholipase and bacteriorhodopsin proteins from lipid bilayers, provides excellent spectral resolution for analytic methods including circular dichroism and analytical ultracentrifugation, and is compatible with standard purification protocols [1]. We have successfully integrated several human GPCRs from eukaryotic membranes including from Pichia, and have found enhanced thermal stability and native-like activity as demonstrated by radioligand binding assays. The Styrene Maleic Acid Lipid Particle (SMALP) method is designed to incorporate protein complexes from cells or membrane fractions into soluble nanoparticles bounded by biocompatible co-polymer without any need for detergent at any stage, offering significantly improved stability, retention of bound lipids as assayed by mass spectrometry, native-like activity. We are developing the SMALP technology as a general solution to preparing native and active membrane proteins for the research community, and for aiding in their structural and functional analysis.

The structural mechanisms underlying Golgi membrane recognition remain unclear, although the targeting domains and role of lipid ligands have been identified. The solution structure of the PH domain of the four-phosphate-adaptor protein has been determined, revealing an exposed hydrophobic wedge that penetrates into mixed micelles designed to mimic the Golgi membrane. Specific recognition of PtdIns^?(4)P and nonspecific engagement of neighbouring phospholipid molecules are mediated through a novel binding mode and an unprecedented burial of hydrophobic bulk. Deep insertion occurs even in the absence of ligand, with a perpendicular orientation of the protein on the micelle based on reduced solvation and paramagnetic relaxation enhancement of embedded backbone and side chain signals. The conservation of key binding features in related PH domains indicates that hydrophobic insertion wedge is a dominant feature of Golgi recognition, and could deform the bilayer as its lipid components are recognized, extracted and trafficked to the cell surface.

References

[1] Knowles, TJ, R Finka, C Smith, YP Lin, T Dafforn, M Overduin, 2009. Membrane Proteins Solubilized Intact in Lipid Containing Nanoparticles Bounded by Styrene Maleic Acid Copolymer J. Am. Chem. Soc. 131, 7484-7485

[2] Cao,X, Ü Coskun, M Rössle, SB Buschhorn, M Grzybek, TR Dafforn, M Lenoir, M Overduin, and K Simons. 2009. Golgi protein FAPP2 tubulates membranes, PNAS online Nov 25.