title: Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA.
abstract: 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 greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' 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 (greater than or equal to 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 A 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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
authors.forename: S C
authors.initials: SC
authors.lastname: Schultz
authors.forename: G C
authors.initials: GC
authors.lastname: Shields
authors.forename: T A
authors.initials: TA
authors.lastname: Steitz
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium; D003460:Crystallization; D002373:Cyclic AMP Receptor Protein / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols; D013096:Spermine; D014961:X-Ray Diffraction
chemical_list: D002373:Cyclic AMP Receptor Protein; D013096:Spermine; D011092:Polyethylene Glycols; D004247:DNA; D002118:Calcium