Improving protein crystal quality by selective removal of a Ca(2+)-dependent membrane-insertion loop - PubMed (original) (raw)

Improving protein crystal quality by selective removal of a Ca(2+)-dependent membrane-insertion loop

David B Neau et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2007.

Abstract

Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca(2+)-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 A crystal structure of 8R-LOX and spectroscopic data suggested that Ca(2+) stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Delta41-45:GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 A improvement in the resolution of the diffraction data.

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Figures

Figure 1

Figure 1

Calcium ions (green) stabilize the membrane-binding loop that contains Trp41–Phe42. Trp41 and Phe42 mediate a packing contact between symmetry mates (pink and blue) in the crystal lattice (PDB code

2fnq

).

Figure 2

Figure 2

Superposition of 8_R_-LOX (gray) on 15-LOX (PDB code

1lox

; blue). The 8_R_-LOX Ca2+-stabilized membrane-insertion loop containing the WF sequence is absent in 15-LOX (Ca2+, green).

References

    1. Boyington, J. C., Gaffney, B. J. & Amzel, L. M. (1997). Adv. Exp. Med. Biol. A, 400, 133–138. - PubMed
    1. Brash, A. R. (1999). J. Biol. Chem.274, 23679–23682. - PubMed
    1. Charron, C., Kern, D. & Giegé, R. (2002). Acta Cryst. D58, 1729–1733. - PubMed
    1. Chen, X. S. & Funk, C. D. (2001). J. Biol. Chem.276, 811–818. - PubMed
    1. Cosme, J. & Johnson, E. F. (2000). J. Biol. Chem.275, 2545–2553. - PubMed

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