PMC:2728246 / 7642-10955 JSONTXT

{"project":"2_test","denotations":[{"id":"19652920-10467150-56190939","span":{"begin":132,"end":136},"obj":"10467150"},{"id":"19652920-15584763-56190941","span":{"begin":554,"end":558},"obj":"15584763"},{"id":"19652920-9835045-56190942","span":{"begin":2297,"end":2301},"obj":"9835045"},{"id":"19652920-9835045-56190956","span":{"begin":3197,"end":3201},"obj":"9835045"}],"text":"Sample and alignment media\nWild-type 15N,13C-labeled human ubiquitin was expressed according to a previous protocol (Johnson et al. 1999) and dispersed in 13 different alignment conditions for RDC measurements. Sample conditions for which datasets 1 through 10 were collected have already been described by (Lakomek et al. 2008a). Datasets 12 and 13 are obtained from additional alignments (positive gel and Pf1 phage) not previously reported.\nSample for dataset 12: A 7% positively charged gel sample was prepared according to (Cierpicki and Bushweller 2004). A stock solution of 40% acrylamide and N,N′-methylenebisacrylamide in a 19:1 ratio was mixed 1:1 with a stock solution of 40% (3-acrylamidopropyl)-trimethylammonium chloride (APTMAC) and N,N′-methylenbisacrylamide in a 19:1 ratio. The mixtures were diluted with 10× TBE buffer (0.9 M TRIS, 0.9 M borate, 0.02 M EDTA, pH = 8.2) to a final concentration of 7%. Polymerization was initiated by the addition of 0.15% ammonium peroxide sulphate and 1% tetramethylethylenediamine. Polymerization of the mixture was carried out overnight in plastic tubes of 3.5 mm in diameter. Gels were extensively washed in deionized water where they swelled significantly to a diameter of 8 mm. Gels were then cut to 4 cm in length to match a ratio between length and diameter of 5:1. Gels were dried for several days on a plastic support wrapped with polyvinylidene chloride foil. To prepare the sample, the dried gel was transferred to a 5-mm microcell tube (Shigemi Inc., Allison Park, PA), ~2 mg of protein were dissolved in 300 μl of buffer (50 mM NaPO4, 100 mM NaCl, pH 6.5, 0.05% NaN3) and added to the gel. The plunger was positioned to limit the final length of the gel to 12 mm.\nSample for dataset 13: The equivalent of 5 mg Pf1 phage (ASLA Ltd., Riga, Lativa) was suspended in buffer (50 mM NaPO4, 100 mM NaCl, pH = 6.5, 0.05% NaN3) and pelleted three times at 140,000 g for 1 h. The pellet was then carefully suspended in a 15N,13C-labeled human ubiquitin sample (~2 mg) in the same buffer. NaCl salt was later added to a final concentration of about 300 mM. The water deuterium quadrupolar splitting was 15.8 Hz.\nAn additional alignment condition and corresponding RDC measurements (dataset 11) were taken from the literature (Ottiger and Bax 1998, 1999). Table 1 shows the corresponding dataset nomenclature.\nTable 1 Summary of sample conditions\nDataset Corresponding SCRM alignment Short description References\n1 A1 7% Positively charged gel (APTMAC/acryl. 1:3) Lakomek et al. (2008a, b)\n2 A2 7% Positively charged gel (APTMAC/acryl. 1:1) Lakomek et al. (2008a, b)\n3 A3 5% Negatively charged gel (acrylic cid/acryl. 1:1) Lakomek et al. (2008a, b)\n4 A4 PEG/hexanol Lakomek et al. (2008a, b)\n5 A7 Bicelles: DMPC/DHPC (3:1) Lakomek et al. (2008a, b)\n6 A8 Bicelles: DMPC/DHPC/SDS (30:10:2) Lakomek et al. (2008a, b)\n7 A9 Bicelles: DLPC/DHPC/SDS (30:10:2) Lakomek et al. (2008a, b)\n8 A10 Bicelles: DMPC/DHPC/C14PC (30:10:1) Lakomek et al. (2008a, b)\n9 A12 Bicelles: DMPC/CHAPSO/CTAB (50:10:1) Lakomek et al. (2008a, b)\n10 A13 Bicelles: DMPC/DHPC/CTAB (30:10:1) Lakomek et al. (2008a, b)\n11 A20 Bicelles: DMPC/DHPC/CTAB (30:10:1) Ottiger and Bax (1998)\n12 – 7% positively charged gel (APTMAC/acryl. 1:1) This work\n13 – Pf1 phages: 15 mg/ml + 300 mM NaCl This work"}