1 / 28

Chaperone-Assisted Crystallography(CAC)

Chaperone-Assisted Crystallography(CAC). Serdar Uysal University of Chicago, Department of Biochemistry and Molecular Biology ISFI. Outline. CAC concept and philosophy Chaperone scaffolds and phage display libraries HTP phage display library sorting CAC for high-hanging fruits.

alaire
Download Presentation

Chaperone-Assisted Crystallography(CAC)

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Chaperone-Assisted Crystallography(CAC) Serdar Uysal University of Chicago, Department of Biochemistry and Molecular Biology ISFI

  2. Outline • CAC concept and philosophy • Chaperone scaffolds and phage display libraries • HTP phage display library sorting • CAC for high-hanging fruits

  3. Chaperone-Assisted Crystallography (CAC) Several different chaperones

  4. Phasing Chaperone

  5. Molecular Scaffolds VH (Gtech) (U of C) (U of C + Gtech)

  6. The Pipeline Chaperone Library Targets Soluble proteins, RNAs, membrane proteins Sorting Chaperone Production Crystallization & Structure Determination of Target-Chaperone Complex

  7. Single Herceptin Fab Scaffold(i.e. no natural scaffold diversity allowed) 1L7I 1ZA3 2H9G 1TZI 1TZH 1S78

  8. Synthetic Fab Libraries X=Y,S,G (50%) and equal mixture of remaining amino acids Why Y,S?

  9. Library Sorting Pipeline 12 Targets in Parallel • • 1H NMR analysis ("simple targets) • Phage binding • • Biotinylation 1 week Target validation and prep automated sorting using a robot (3 rounds); Confirm success of sorting 1 weeks Transfer a mixture of clones to expression vector 1 week HTP protein production and BIAcore assay • Expression level • Affinity 1 week 6 chaperones for each target Total: 4 weeks

  10. Fab clones for MCSG targets Targets provided by H. Li, F. Collart, and A. Joachimiak (MCSG)

  11. Benchmarking the Fab CAC pipeline with 17 MCSG Targets Fellouse et al. JMB, in press

  12. Successful Examples of CAC for High Hanging Fruits

  13. Crystal structureof FAB2-C209 (1.95 Å) Buried surface area: 1316 Å2 Jingdong Ye, Valya Tereshko, Joe Piccirilli

  14. KcsA with Fab fragment Crystal lattice of the KcsA+ channel in complex with a proteolytic FAB fragment (PDB code 1k4c)

  15. KcsA ? 8 binders produced

  16. Res=3.7A° Rwork=34% Rfree=28%

  17. Reinterpretation of Closed Conformation

  18. Packing and Challenges

  19. C-terminal domain at 2.6A° 2X Binding Fab_10 4X Binding Fab_2

  20. 4-Helix Bundle C-terminal Domain

  21. Conclusions • Synthetic libraries based on restricted AA diversity. • HTP pipeline for chaperone engineering. • High-affinity chaperones for membrane proteins, functional RNAs and protein complexes. • Novel structures using the CAC strategy.

  22. The Univ of Chicago Tony Kossiakoff Shohei Koide Magda Bukowska Vince Cancasci Kaori Esaki Ryan Gilbreth James Horn Akiko Koide Brenda Leung James Raptis Valya Tereshko John Wojcik Jingdong Ye Valeria Vazquez Joe Piccirilli Eduardo Perozo Acknowledgments Genentech, Inc. Dev Sidhu Fred Fellouse Sara Birtalan Pierre Barthelemy UC Core Facilities Flow Cytometry Facility DNA sequencing Facility Advanced Photon Source/ANL Funding: NIH (NIGMS, NIDDK), HHMI, UC Cancer Research Center

  23. Tertiary complex

More Related