W0245

A Solubility Optimization Screen and the Crystallization of Aeropyrum pernix Flap Endonuclease-1. Brandon K. Collins¹, Mike W. Kaiser², and Timothy C. Mueser¹, ¹Dept. of Chemistry, The Univ. of Toledo, Toledo, OH 43606, ²Third Wave Technologies, Inc., Madison, WI 53719.

Crystallization of protein and protein complexes is a multi-parametric problem that involves investigation of a vast number of physical and chemical conditions. The buffer, salts, and additives used to prepare the protein will be present in every crystallization condition. It is imperative that these conditions be defined prior to crystal screening since they will have a ubiquitous involvement in the crystal growth experiments. Our study involves the crystallization of flap endonuclease (FEN-1) DNA repair enzymes from five different species of Archaea. Using a solubility screen, we were able to dramatically increase the maximum solubility of the proteins prior to crystal screens. We have observed a significant correlation between maximizing the solubility of a protein with positive results in crystal screens. Optimum solubility parameters were obtained using an adaptation of the “ion screen” (Mueser et al. Biochemistry, 2000, V39, pp. 15353 - 64). The “ion screen” is the precursor to the PEG/Ion Screen (Bob Cudney, Hampton Research, personal communication). Crystallization conditions were screened using a 96 well format Corning crystal trays, involving ten 48 condition crystal kits at 4ºC and 22ºC (960 conditions total per enzyme). Proteins were dialyzed into specific solvent conditions, formulated using results from the solubility screen. The FEN-1 from Aeropyrum pernix (Ape) yielded large diffraction quality crystals directly from our initial screens. Crystal screen results from Ape FEN-1 dialyzed into standard chromatography buffer conditions were compared with the results from the solubility optimized screen. The solubility optimized protein produced large diffraction quality crystals under multiple conditions in which the non-optimized protein produced only precipitate. The Ape FEN-1 crystallized in space group P61 with diffraction observed to 1.4Å resolution. Large solvent channels are observed in the lattice and active site metal exchange experiments have been conducted. The refined structure is presented elsewhere (see Stephen Tomanichek et al. this meeting).