Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase

NJ Gunn; MA Gorman; RCJ Dobson; MW Parker; TD Mulhern

Journal article

Purification, crystallization, small-angle X-ray scattering and preliminary X-ray diffraction analysis of the SH2 domain of the Csk-homologous kinase

NJ Gunn, MA Gorman, RCJ Dobson, MW Parker, TD Mulhern

Acta Crystallographica Section F Structural Biology and Crystallization Communications | INT UNION CRYSTALLOGRAPHY | Published : 2011

DOI: 10.1107/S1744309110053728

Abstract

The C-terminal Src kinase (Csk) and Csk-homologous kinase (CHK) are endogenous inhibitors of the proto-oncogenic Src family of protein tyrosine kinases (SFKs). Phosphotyrosyl peptide binding to their Src-homology 2 (SH2) domains activates Csk and CHK, enhancing their ability to suppress SFK signalling; however, the detailed mechanistic basis of this activation event is unclear. The CHK SH2 was expressed in Escherichia coli and the purified protein was characterized as monomeric by synchrotron small-angle X-ray scattering in-line with size-exclusion chromatography. The CHK SH2 crystallized in 0.2 M sodium bromide, 0.1 M bis-Tris propane pH 6.5 and 20% polyethylene glycol 3350 and the best cry..

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University of Melbourne Researchers

Michael Gorman Author

Renwick Dobson Author

Michael Parker Author

Terry Mulhern Author

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Grants

Awarded by Australian National Health and Medical Research Council (NHMRC)

Awarded by Australian Institute of Nuclear Science and Engineering (AINSE)

Funding Acknowledgements

We would like to acknowledge the support and assistance of the staff at the Bio21 Collaborative Crystallographic Centre at CSIRO Molecular and Health Technologies, Parkville, Melbourne. This research was undertaken on the MX2 and SAXS/WAXS beamlines at the Australian Synchrotron, Victoria, Australia; we would like to thank Tom Caradoc-Davies and Nigel Kirby, respectively, for helpful discussion and assistance at the Australian Synchrotron. This work was supported by Australian National Health and Medical Research Council (NHMRC) Project Grant 509115 and Australian Institute of Nuclear Science and Engineering (AINSE) Award 09137 to TDM. RCJD acknowledges the C. R. Roper Fellowship for support. MWP is an Australian Research Council Federation Fellow and NHMRC Honorary Fellow.