Structural and functional analysis of cell-free expressed CCR5, a coreceptor required for HIV infection; and establishment of a new detergent free reconstitution method.
Zusammenfassung der Projektergebnisse
Successful functional and structural analysis of cell-free expressed CCR5 in solution requires the presence of detergents. Unfortunately, the presence of all tested detergents prevented any functional studies of CCR5 by surface plasmon resonance (SPR) and by NMR titration with its ligand CCL14. Besides the characterization of CCR5, my proposed research aimed on the establishment of a new detergent free reconstitution method. As the proposed cell-free integral membrane protein (IMP) expression into liposomes as well as into nonodiscs was successfully shown by other groups in early 2008, I changed my focus on addressing another very important problem in cell-free expression of G-protein coupled receptors (GPCRs): aggregation and sample homogeneity in the presence of detergents. Although cell-free expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. In my resulting studies, I could show that using cell-free expression with the help of a fructose-based polymer, NV10 polymer (NVoy), I obtained preparative amounts of homogenous GPCRs from the three GPCR families. I could demonstrate that two GPCR B family members, corticotrophinreleasing factor receptor 1 and 2 are not only solubilized in NVoy but also have functional ligand-binding characteristics with different antagonists in a detergentfree environment as well. This study showed the first cell-free expression of members of all three GPCR families and demonstrates ligand binding for B family GPCRs. The use of NVoy allows to overcome detergent related aggregation problems and enabled the production of non-aggregated high quality samples from the precipitate cell-free (P-CF) mode as well as with the newly introduces soluble NVoy cell-free (SN-CF) mode. Cell-free systems are still a new avenue for expressing IMPs, but I showed its applicability to difficult IMPs, such as GPCRs, which can now be synthesized in preparative amounts. Now, the use of nondetergent-like polymers may improve protein stability and maintain the protein’s function, thereby enabling new possibilities for functional and structural studies of GPCRs and difficult to obtain IMPs in general.
Projektbezogene Publikationen (Auswahl)
- Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis. Proc. Natl. Acad. Sci. USA, 107, 10902-7. (2010)
I. Maslennikov, C. Klammt, E. Hwang, G. Kefala, M. Okamura, L. Esquivies, K. Mörs, C. Glaubitz, W. Kwiatkowski, Y. H. Jeon, and S. Choe
- Polymer-based cell-free expression of ligand binding family B G-protein coupled receptors without detergents. Protein Sci., 20, 1030-41. (2011)
C. Klammt, M.H. Perrin, I. Maslennikov, L. Renault, M. Krupa, W. Kwiatkowski, H. Stahlberg, W. Vale, and S. Choe
