A biosensor-based approach toward purification and crystallization of G protein-coupled receptors

Analytical Biochemistry
Iva NavratilovaDavid G Myszka

Abstract

Biacore technology was used to develop an affinity purification method and screen cocrystallization conditions for the chemokine receptor CCR5. We characterized the binding of nine HIV gp120 variants and identified a truncated construct (YU2DV1V2) that bound CCR5 independent of CD4. This construct was used in an affinity purification step to improve the activity of detergent-solubilized receptor by approximately 300%. The biosensor was also used to screen receptor binding activity automatically under 50 different crystallization conditions. We found that high-molecular-weight polyethylene glycols (PEGs 4,000 and 8,000 Da) most often stabilized the receptor and improved complex formation with potential cocrystallization partners such as conformationally sensitive monoclonal antibodies and gp120. Our results show how biosensors can provide unique insights into receptor purification methods and reveal the effects of crystallization conditions on complex formation. Importantly, these methods can be readily applied to other systems.

References

Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·D D OprianH G Khorana
Sep 25, 1999·The Journal of Biological Chemistry·T MirzabekovJ Sodroski
Apr 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·T DragicJ P Moore
Aug 2, 2000·Proceedings of the National Academy of Sciences of the United States of America·D G MyszkaM L Doyle
Aug 5, 2000·Science·K PalczewskiM Miyano
Mar 31, 2005·Analytical Biochemistry·Iva NavratilovaDavid G Myszka

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Citations

Dec 14, 2012·Nature·Francis AlonzoVictor J Torres
Oct 19, 2011·Future Medicinal Chemistry·Iva Navratilova, Andrew L Hopkins
Jan 13, 2009·Analytical Biochemistry·Rebecca L RichDavid G Myszka
Jun 30, 2007·Journal of Internal Medicine·S PhogatG B Karlsson Hedestam
Jun 10, 2006·Analytical Biochemistry·Iva NavratilovaDavid G Myszka
Oct 26, 2010·Analytical Biochemistry·Rebecca L RichDavid G Myszka
Jun 20, 2013·Biochimica Et Biophysica Acta·Silvia C Locatelli-HoopsAlexei A Yeliseev
Dec 13, 2007·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Nov 10, 2011·Journal of Biomolecular Screening·Iva NavratilovaAndrew Cook
Apr 29, 2010·Journal of the American Chemical Society·Chie YoshiuraIchio Shimada
Jul 19, 2011·ACS Medicinal Chemistry Letters·Iva NavratilovaAndrew L Hopkins
Jan 24, 2014·ACS Medicinal Chemistry Letters·Tonia AristotelousIva Navratilova

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