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Simon, B.* ; Madl, T. ; Mackereth, C.D.* ; Nilges, M.* ; Sattler, M.

An efficient protocol for NMR-spectroscopy-based structure determination of protein complexes in solution.

Angew. Chem.-Int. Edit. 49, 1967-1970 (2010)

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Open Access Green möglich sobald Postprint bei der ZB eingereicht worden ist.

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Sample preparation. A construct containing U2AF65 residues 148 to 342 (RRM12) was cloned into a modified pET9d vector containing an N-terminal His6-tag followed by a TEV protease cleavage site. Site-specific cysteine mutants were created individually at positions 155, 164, 171, 187, 188, 209, 273, 281, 287 and 318 by PCR amplification with overlapping oligonucleotides containing the mutated sequence, and afterwards verified by sequencing. Wild type and mutant U2AF65(RRM12) were produced in BL21(DE3) or BL21(DE3)pLysS cells using minimal M9T media supplemented with 2 g l-1 [13C]-glucose and/or 1 g l-1 [15N]- ammonium chloride. Following cell lysis, the proteins are purified by Ni2+ affinity chromatography resin in a buffer containing 50 mM Tris (pH 7.5), 500 mM NaCl, 5 % (v/v) glycerol with imidazole concentrations of 5, 30 and 250 mM for binding, wash and elution, respectively. The buffer was exchanged to phosphate buffered saline prior to removal of the His6-tag with 20 μg l-1 TEV protease. The His6-tag, uncleaved protein and TEV protease werre removed by Ni2+ affinity chromatography, and the final sample placed into 20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 0.1% sodium azide and 1 mM EDTA. To make the samples for PRE measurement, the protein was completely reduced by the addition of 2 mM dithiothreitol, and extensively dialyzed in 50 mM Tris (pH 8.0) and 200 mM NaCl. Three molar equivalents of methanol-dissolved 3-(2-iodoacetamido)-2,2,5,5,tetramethyl-1- pyrrolidinyloxy radical (iodoacetamido-PROXYL; Sigma-Aldrich) was added and the reaction left in the dark at +4 °C for 16 hours. Unreacted spin label was removed following three changes of buffer into 20 mM sodium phosphate (pH 6.5), 50 mM NaCl, 0.1% sodium azide and 1 mM EDTA using a PD10 desalting column (GE Healthcare Life Sciences). The U9 RNA oligonucleotide was purchased from Biospring GmbH, Frankfurt (Germany) and dissolved into water.

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