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Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization
JoVE Journal
Bioquímica
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JoVE Journal Bioquímica
Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

DOI:
10.3791/60747-v

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09:19 min

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March 16, 2020

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, , , , , ,
1Laboratory of Biomolecular Research,Paul Scherrer Institute, 2Department of Biology,ETH Zürich, 3Center for Radiopharmaceutical Sciences,Paul Scherrer Institute, 4Laboratory of Molecular Biology,Medical Research Council

Capítulos

  • 00:04Introduction
  • 00:45Cell Membrane Solubilization and Protein Extraction
  • 03:53UV-VIS Spectroscopy
  • 04:33Automated Size-Exclusion Chromatography of Rhodopsin and Rhodopsin Mini-Go Complex
  • 06:04Deglycosylation
  • 06:44Results: UV-VIS Spectroscopy, SEC, and SDS-PAGE
  • 08:34Conclusion

Summary

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This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.

Tags

GPCRMini-G ProteinSignal TransductionMembrane Protein ComplexDetergent ScreeningCrystallizationHEK293 CellsAffinity Purification9-cis RetinalBuffer Exchange

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