Abstract
High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1–7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (http://www.nigms.nih.gov/psi). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.
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J.H.M. Cabral A. Lee S.L. Cohen B.T. Chait M. Li R. Mackinnon (1998) Cell 95 649–655 Occurrence Handle10.1016/S0092-8674(00)81635-9 Occurrence Handle9845367
S.L. Cohen A.R. Ferré-D’Amaré S.K. Burley B.T. Chait (1995) Protein Sci. 4 1088–1099 Occurrence Handle7549873
S.L. Cohen B. Chait (2001) Annu. Rev. Biophys. Biomol. Struct. 30 67–85 Occurrence Handle10.1146/annurev.biophys.30.1.67 Occurrence Handle11340052
J. Marcotrigiano A.C. Gingras N. Sonenberg S.K. Burley (1997) Cell 89 951–961 Occurrence Handle10.1016/S0092-8674(00)80280-9 Occurrence Handle9200613
C.H. Lee K. Saksela U.A. Mirza B.T. Chait J. Kuriyan (1996) Cell 85 931–942 Occurrence Handle10.1016/S0092-8674(00)81276-3 Occurrence Handle8681387
X. Xie T. Kokubo S.L. Cohen U.A. Mirza A. Hoffmann B.T. Chait R.G. Roeder Y. Nakatani S.K. Burley (1996) Nature 28 316–322 Occurrence Handle10.1038/380316a0
G. Zhang Y. Liu J. Qin B. Vo W. Tang A.E. Ruoho J.H. Hurley (1997) Protein Sci. 6 903–908 Occurrence Handle9098900
M. Cadene B. Chait (2000) Anal. Chem. 22 5655–5658 Occurrence Handle10.1021/ac000811l
A. Bateman L. Coin R. Durbin R.D. Finn V. Hollich S. Griffiths-Jones A. Khanna M. Marshall S. Moxon E.L. Sonnhammer D.J. Studholme C. Yeats S.R. Eddy (2004) Nucleic Acids Res. 32 D138–141 Occurrence Handle10.1093/nar/gkh121 Occurrence Handle14681378
D.T. Jones (1999) J. Mol. Biol. 292 195–202 Occurrence Handle10.1006/jmbi.1999.3091 Occurrence Handle10493868
S.F. Altschul T.L. Madden A.A. Schaffer J. Zhang Z. Zhang W. Miller D.J. Lipman (1997) Nucleic Acids Res. 25 3389–3402 Occurrence Handle10.1093/nar/25.17.3389 Occurrence Handle9254694
K.M. Kim E.C. Yi D. Baker K.Y.J. Zhang (2001) Acta Crystallogr. D57 759–762
M.H. Bucher A.G. Evdokimov D.S. Waugh (2002) Acta Crystallogr. D58 392–397
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Gao, X., Bain, K., Bonanno, J.B. et al. High-throughput Limited Proteolysis/Mass Spectrometry for Protein Domain Elucidation. J Struct Funct Genomics 6, 129–134 (2005). https://doi.org/10.1007/s10969-005-1918-5
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DOI: https://doi.org/10.1007/s10969-005-1918-5