Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site: The use of modular DNA

ACS Citation

Schultz, S. C.; Shields, G. C.; Steitz, T. A. Crystallization of Escherichia coli Catabolite Gene Activator Protein with Its DNA Binding Site: The Use of Modular DNA. J. Mol. Biol. 1990, 213 (1), 159-€“166.


To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments ≥28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5-² or 3-² termini. In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (≥28 bp) required by this protein. We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 {\AA} resolution. Additives such as calcium, n-octyl-$\beta$-d-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III. Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol.

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Journal of Molecular Biology

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