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A major focus of the MPSBC project is the development of technologies to reduce the time and effort required for a TMP target to progress from target identification and cloning to the production of well-diffracting crystals.  The papers referenced here describe recent technology-development work by the MPSBC PIs.

 

PURIFICATION OF TRANSMEMBRANE PROTEINS

dumontDUMONT LAB
To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents. [ Learn More ]

PRE-CRYSTALLIZATION SCREENING

wienerWIENER LAB
Structural studies on integral membrane proteins are routinely performed on protein-detergent complexes (PDCs) consisting of purified protein solubilized in a particular detergent. Of all the membrane protein crystal structures solved to date, a subset of only four detergents has been used in more than half of these structures. Unfortunately, many membrane proteins are not well behaved in these four detergents and/or fail to yield well-diffracting crystals. Identification of detergents that maintain the solubility and stability of a membrane protein is a critical step and can be a lengthy and "protein-expensive" process. We have developed an assay that characterizes the stability and size of membrane proteins exchanged into a panel of 94 commercially available and chemically diverse detergents. This differential filtration assay (DFA), using a set of filtered microplates, requires sub-milligram quantities of purified protein and small quantities of detergents and other reagents and is performed in its entirety in several hours. [ Learn More ]

HIGH-THROUGHPUT MEMBRANE PROTEIN CRYSTALLIZATION SCREENS

malkowskiMALKOWSKI LAB
One of the goals of the Membrane Protein Structural Biology Consortium (MPSBC) is to develop crystallization screens specifically tailored to membrane proteins. 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 design crystallization screens rationally. Dye-based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents have been performed 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. Both the experimentally derived phase boundary data and this membrane screen are now available through the high-throughput crystallization facility located at the Hauptman-Woodward Institute. (http://www.mpsbc.org/About/HTS_Services/hts_services.html) In addition, the phase boundary data have been packaged into an interactive Excel spreadsheet (SlickSpot.xls) that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. [ Learn More]

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