Polymer-basierte Nanodisks zur Untersuchung der Struktur und Dynamik von G-protein-gekoppelten Rezeptoren und anderen klinisch relevanten Membranproteinen
Zusammenfassung der Projektergebnisse
Three-dimensional structures of membrane proteins are of paramount value for understanding protein function on a molecular level. However, membrane-protein structure determination is a continuing bottleneck in the field of protein crystallography. For some membrane proteins, detergent-mediated solubilization compromises protein stability and functionality, often impairing biophysical and structural analyses. As an alternative to approaches mediated by conventional detergents, we report the crystallogenesis of a recombinantly produced membrane protein that never left a lipid bilayer environment. We used styrene–maleic acid (SMA) copolymers to solubilize lipid-embedded proteins into SMA nanodiscs, purified these discs by affinity and size-exclusion chromatography, and transferred proteins into the lipidic cubic phase (LCP) for in meso crystallization. The 2.0-Å structure of an a-helical 7-transmembrane microbial rhodopsin thus obtained is of high quality and virtually identical to the 2.2-Å structure obtained from traditional detergent-based purification and subsequent LCP crystallization. In addition, this allowed us to report that SMA copolymers are compatible with LCPs and that lipid nanodiscs enable transfer of membrane proteins between lipid-bilayer systems. Taken together, this work expands the toolbox for membrane-protein crystallography and has been received equally enthusiastically by structural biologists, polymer scientists, and membrane-protein (bio)chemists with an academic or industrial background at more than 10 oral presentations in Europe and North America. It was also recognized by the public through an interview with Milka Kostic from Structure’s Biology in 3D blog (www.crosstalk.cell.com) and through several press releases as well as various tweets. Finally, with the polymer community growing rapidly, we and others founded the SMALP network (www.smalp.net), and organized the North American SMALP Meetings. Membrane-protein structure determination is also compromised by the difficulties associated with harvesting small membrane-protein crystals, which is why, in situ data collection—where crystals are not harvested and flash-frozen but placed in the X-ray beam within their growth medium—has been a major focus of progress in protein crystallography. In a second project, we introduced the Mylar in situ method using Mylar-based sandwich plates that are inexpensive, easy to make and handle, and show significantly less background scattering than other setups. A variety of cognate holders for patches of Mylar in situ sandwich films corresponding to one or more wells makes the method robust and versatile, allows for storage and shipping of entire wells, and enables automated crystal imaging, screening, and goniometer-based X-ray diffraction data-collection at room temperature and under cryogenic conditions for soluble and membrane-protein crystals grown in or transferred to these plates. This approach promises high-resolution structural studies of membrane proteins to become faster and more routine and, therefore, has become highly popular among our co-authors and collaborators in a variety of scientific fields. It is currently used to grow novel GPCR crystals in situ as well as to develop in situ solutions for serial crystallography at, respectively, synchrotrons and free electron lasers. The University of Toronto is in the process of licensing the technology in order to make plates and holders commercially available soon. The Mylar in situ approach was received enthusiastically at more than 10 invited oral presentations in North America and Europe. As judged from the feedback we received, there is considerable interest in cutting-edge in situ technology, which is why we just published a comprehensive state-of-the-art step-by-step protocol that serves as a reference. Finally, in a third project I co-authored a paper on the use of microfluidics in membrane-protein crystallography. Our work was also recognized by the public through an article in Noteworthy in 2016 and a publication covering both main projects in Biospektrum in 2017.
Projektbezogene Publikationen (Auswahl)
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(2016) A Versatile System for High-Throughput In Situ X-ray Screening and Data Collection of Soluble and Membrane-Protein Crystals. Crystal growth & design 16 (11) 6318–6326
Broecker, J., Ernst, O. P. et al.
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(2018) High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions. Nature protocols 13 (2) 260–292
Broecker, Jana; Morizumi, Takefumi; Ou, Wei-Lin; Klingel, Viviane; Kuo, Anling; Kissick, David J.; Ishchenko, Andrii; Lee, Ming-Yue; Xu, Shenglan; Makarov, Oleg; Cherezov, Vadim; Ogata, Craig M.; Ernst, Oliver P.
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A versatile system for high-throughput in situ X-ray screening and data collection of soluble and membrane-protein crystals. Cryst. Growth Des. 16, 6318– 6326 (2016)
Broecker J., Klingel V., Ou W.-L., Balo A. R., Kissick D. J., Ogata C. M., Kuo A., Ernst O. P
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Crystallogenesis of membrane proteins mediated by polymer-bounded lipid nanodiscs. Structure 25, 384–392 (2017)
Broecker, J.; Eger, B. T.; Ernst, O. P.
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Vom Einsatz polymerbasierter Lipidnanodiscs sowie insitu-Methoden. Biospektrum 3, 267–269 (2017)
Broecker J., Ernst, O. P.
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X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization. Biomicrofluid. 11, 024118-1–024118-13 (2017)
Schieferstein, J. M., Pawate, A. S., Sun, C., Wan, F., Sheraden, P. N., Broecker, J., Ernst, O. P., Gennis, R. B., Kenis, P. J. A.
