Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins

Abstract

Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein-detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye-based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536-cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high-throughput crystallization facility located at the Hauptman-Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X-ray diffraction.

Substrate-selective Inhibition of Cyclooxygeanse-2 by Fenamic Acid Derivatives Is Dependent on Peroxide Tone

“…As such, we utilized the 1536 condition tailored membrane protein screen (32) in the HighThroughput Crystallization Laboratory at the HauptmanWoodward Institute (33) to search for new leads that would facilitate crystallization. Although numerous crystallization leads were identified, one lead was successfully optimized and converted from microbatch-under-oil format to the sitting drop vapor-diffusion format.…”

The detection and subsequent volume optimization of biological nanocrystals

“…The first set, designed to identify initial crystallization conditions of soluble proteins, is comprised of 688 in-house cocktails based on an incomplete-factorial sampling of salts, buffers, and polymers and 848 chemically complementary cocktails from Hampton Research crystallization screens (Aliso Viejo, CA); cocktails are annually reviewed and refined 8 . A second set of 1536 cocktails designed to crystallize membrane protein-detergent complexes, grid-samples chemical space near detergent phase boundaries, 15 where the crystallization is more likely to occur 16 . The set of 60 proteins used in this study was not random.…”

The Transition from Closed to Open Conformation of Treponema pallidum Outer Membrane-associated Lipoprotein TP0453 Involves Membrane Sensing and Integration by Two Amphipathic Helices

“…Crystallization and Data Collection-Initial crystallization screening was carried out in the High Throughput Crystallization Laboratory at the Hauptman-Woodward Medical Research Institute utilizing a sparse-matrix screen designed for soluble proteins and a tailored grid screen optimized for membrane proteins that is based on detergent phase partitioning (23,24). A 1536 mixture screen utilizing the microbatch-underoil method was set up with purified TP0453 in 25 mM Tris (pH 7.2), 150 mM NaCl containing 0.15% (w/v) n-octyl-␤-D-glucopyranoside (␤-OG) at a protein concentration of 8.2 mg/ml.…”