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Published January 14, 2014 | Supplemental Material + Published
Journal Article Open

Self-assembled lipid and membrane protein polyhedral nanoparticles

Abstract

We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation ofMPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics- based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation.

Additional Information

© 2014 National Academy of Sciences. Contributed by Douglas C. Rees, November 26, 2013 (sent for review January 28, 2012). We thank our colleagues at the University of Colorado and California Institute of Technology (Caltech) for helpful comments and criticisms, Randal Bass (Caltech) for providing initial MscS samples, Mark Yeager and Brad Bennett (University of Virginia) for providing samples of Cx26, Steven Ludtke (Baylor University) for EMAN advice, Rob Phillips and Christoph Haselwandter (Caltech) for discussions on polyhedral symmetry, and Chris Arthur (Oregon Health and Science University) and Axel Brilot (Brandeis University) for assistance with image scanning. Molecular graphics images were produced using the University of California, San Francisco (UCSF) Chimera package from the Resource for Biocomputing, Visualization, and Informatics at UCSF (supported by NIH-P41RR001081). Electron microscopy was performed in the Laboratory for 3D Electron Microscopy of Cells at the University of Colorado (supported by NIH-P41GM103431-42). This work was supported in part by a National Institutes of Health (NIH) Exceptional, Unconventional Research Enabling Knowledge Acceleration (EUREKA) Award (to M.H.B.S.), a Howard Hughes Medical Institute Collaborative Innovation Award (to D.C.R. and M.H.B.S.), and NIH Grant GM084211 (to D.C.R.). Author contributions: T.B., H.-J.W., M.K.M., Y.C.L., D.C.R., and M.H.B.S. designed research; T.B., H.-J.W., M.K.M., J. Lee, N.G., J. Lai, K.W., D.C.R., and M.H.B.S. performed research; J. Lee, J. Lai, J.M.H., K.W., and Y.C.L. contributed new reagents/analytic tools; T.B., H.-J.W., M.K.M., J. Lee, N.G., J.M.H., Y.C.L., D.C.R., and M.H.B.S. analyzed data; and D.C.R. and M.H.B.S. wrote the paper.

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Published - PNAS-2014-Basta-670-4.pdf

Supplemental Material - pnas.201321936SI.pdf

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