Louis, MO) unless otherwise indicated

Louis, MO) unless otherwise indicated. binding website. The four domains indicated in b were indicated as His/Xpress-tagged proteins, purified, and subjected to SDS-PAGE and Western blotting. Domain 1 consists of amino acids 1C119, website 2 contains amino acids 126C214, website 3 contains amino acids 210C413, and website 3 + 4 consists of amino acids 210C441. Blots were probed either with anti-Xpress, to show the positions of the proteins, or with the GST-GGA1 appendage construct. The website 3 + 4 create was indicated at lower levels than the additional constructs. Website 1 binds to the GGA appendage. Notice the higher molecular mass band in the lane containing website 3 (asterisk), which may be an SDS-resistant homodimer. (f) Peptide competition for binding. A 15-residue peptide derived from the NH2-terminal website of the common isoform of p56, DDDDFGGFEAAETFD, was added to the incubation mixtures. Top, a blot of website 1 was probed with the GST-GGA1 appendage create with increasing concentrations of peptide. Bottom, the GST-GGA1 appendage create was used to pull down proteins from pig mind cytosol with increasing concentrations of peptide, and a European blot of the pulldowns was probed with anti-p56. In both cases, competition is essentially total at 0.5 mM peptide. (g) Cross-linking of website 3. Website 3 was run on a nonreducing gel, either with or without 1st treating with the cross-linker DSP, and the Western blot was probed with anti-Xpress. The anomalous mobility of the noncross-linked MK-5172 hydrate band is definitely presumably due to the gel system. The band in the cross-linked lane is approximately twice the apparent molecular mass of the band in the noncross-linked lane, suggesting that it has been cross-linked into a homodimer. (h) Model of p56. The protein is predicted to form a coiled coil homodimer by using domains 2 and 3. The NH2-terminal website (website 1) interacts with the GGA appendage domains. The COOH-terminal website (website 4), which is the most conserved part of the protein, may interact with another binding partner(s). Antibodies Antibodies were raised in rabbits against the two novel proteins recognized with this study, p200 and p56. For the p200 antibodies, clone KIAA1414 (generously provided by the Kazusa DNA Study Institute, Chiba, Japan) was used as a template to amplify the coding sequence for amino acids 946-1217 by PCR, and the product was ligated into pGEX4T-1 (Amersham Biosciences, Piscataway, NJ). The producing GST fusion protein was soluble and was purified as specified by the manufacturer. Rabbits were immunized with the GST-p200 construct and with the His-p56 construct described above, and the antisera were affinity purified as explained previously (Page with an NH2-terminal 6xHis tag. The protein was purified by Ni-nitrilotriacetic acid affinity chromatography, followed by gel filtration in 5 mM HEPES (pH 7.5), 100 mM NaCl. The protein was concentrated to 37 mg/ml and crystallized by sitting drop vapor diffusion in 100 mM sodium citrate (pH 5.6), 1.7 M ammonium sulfate. Crystals grew to maximum dimensions of 1 1 0.8 0.25 mm within 24 h and belong to space group P3221 with unit cell dimensions a = 65.4 ?, b = 65.4 ?, c = 142.7 ?, = = 90, and = 120. In our crystals, we observe an extensive interface between two individual GGA1 proteins in the asymmetric unit; however, we do not see any evidence for dimerization in solution by using either gel filtration MK-5172 hydrate or dynamic light scattering, suggesting that this is not an in vivo natural dimeric conversation. Data Collection and Structure Determination Crystals were mounted in mother liquor PSFL made up of 20% (vol/vol) glycerol, and data collected at 100 K by using a Rigaku rotating anode x-ray source fitted with a MAR345 image plate detector. A data set was collected to MK-5172 hydrate 2.3-? resolution, integrated with MOSFLM (Leslie, 1992 ), and scaled using CCP4 programs (Dodson = _(Fp-Fcalc)/_Fp eFrom PROCHECK (Laskowski and decided its structure by x-ray crystallography. Crystals of the GGA1 appendage diffract to better than 2.3-? resolution, and the structure was solved by molecular replacement using the appendage structure as a starting model (Kent contains no other obvious homologues of p200, there may be another protein or proteins that can perform the same role. -Synergin is the only one of the three shared binding partners that had already been identified and characterized. In our previous study, we showed by Western blotting that -synergin could MK-5172 hydrate be pulled down by both and GGA appendages (Hirst em et al. /em , 2000 ). We have now confirmed this result by.