W0209

"How Does a Low-Melting Organic Salt Replace the Hydration Shell of a Protein?" Jennifer A. Garlitz, Catherine Summers, Robert A. Flowers II and Gloria E. O. Borgstahl, The University of Toledo, Department of Chemistry, Toledo, OH 43606

Life as we know it requires large amounts of water which plays a fundamental role in the biochemical processes of enzyme activity, protein stability and protein crystallization. Paradoxically, lyophilized enzymes dissolved in neat low melting organic salts (LMOS) have been shown to retain enzymatic activity and to have higher thermal stability than their aqueous counterpart. Therefore, protein crystals grown from solutions of LMOS may be of higher diffraction quality and more robust than those grown from aqueous solutions. A series of LMOS have been synthesized and characterized which have water-like properties, including remaining a clear liquid at room temperature and containing hydrogen bond acceptor and donor atoms. In addition, LMOS have hydrophobic and ionic character and some proteins and some substrate/protein complexes may crystallize from solutions containing LMOS that are simply not possible to crystallize from aqueous solutions. Also, the industrial application of enzymes to perform organic reactions would be improved by the exclusion of water from the system. The crystal structure of a protein in the presence of a LMOS instead of water has been investigated to provide structural information about how the salt interacts with the protein and replaces the interactions of water.

Our current studies focus on the LMOS ethylammonium nitrate (EAN). Lysozyme has been shown to be soluble in EAN to >200 mg/ml and to retain approximately 75% of its activity. In order to provide a structure-based understanding of how EAN intimately interacts with proteins and their surrounding solvent we have performed several crystal soaking experiments. Tetragonal lysozyme crystals were grown to a uniform in size (approximately 250 mm³) and crosslinked with 0.75% glutaraldehyde. The glutaraldehyde solution was replaced by buffered 20% NaCl and the crystals were allowed to soak for 6 h. Here and at each additional soaking step two crystals were reserved for data collection. Sequential soaks of increasing EAN were performed with a 10% stepsize. X-ray diffraction data were collected to 1.8 Å resolution on crystals soaked in 20, 30 and 40% EAN with an MSC R-AXIS IV imaging plate system equipped with CuK_[alpha] radiation from a rotating anode X-ray generator. R_merge statistics indicate differences ranging from 16 to 22%. For each 10% of EAN soaked into the crystals the differences increase by 6.0-7.4%. Preliminary electron density maps indicate that EAN binding sites will be identified from the 40% EAN soaked crystals. Refinement of the solvent structure and analysis of the interaction of EAN with lysozyme will be presented. In addition, progress towards the crystallization of lyophilized lysozyme dissolved in neat EAN will also be reported.