A monovalent ligand binds the lattice by interacting with several consecutive models. antibody (MW1) Fab Rabbit Polyclonal to CD91 fragment, which recognizes full-length huntingtin in mouse brain sections, binds specifically to exon 1 constructs made up of normal and expanded poly(Gln) tracts, with affinity and stoichiometry that increase with poly(Gln) length. These data support a linear lattice model for poly(Gln), in which expanded poly(Gln) tracts have an increased quantity of ligand-binding sites as compared with normal poly(Gln). The linear lattice model provides a rationale for pathogenicity Methazathioprine of expanded poly(Gln) tracts and a structural framework for drug design. Experimental studies of polyglutamine [poly(Gln)] have historically been hampered by insolubility, and molecular modeling, biophysical characterization of solubilized peptides, and antibody-binding studies have provided conflicting results as to its structure. Theoretical work suggested either random coil or more ordered structures (e.g., a -hairpin) (examined in ref. 1). Circular dichroism (CD) studies of normal poly(Gln) peptides (Q9 or Q17 flanked by -helical peptides) (2) revealed random-coil structures, as did normal and expanded poly(Gln) peptides (K2Q= 5C49) in aqueous answer after a disaggregation process involving nonnative solvents (3). A recent NMR study of normal and expanded poly(Gln) fused to glutathione TRX. Inserts made up of HD exon 1 with a C-terminal His6 tag produced by restriction enzyme digestions or PCR amplification (requiring the addition of 10% dimethyl sulfoxide or 10% glycerol) were subcloned into the for 10 min at 4C, and pellets were resuspended in 15 mM Tris?HCl, pH 8.0, and incubated 45 min with shaking at 4C. Osmotically shocked cells were spun at 15,000 for 10 min at 4C, and the supernatant made up of the protein of interest was incubated with Ni nitrilotriacetate beads (Qiagen). Proteins were eluted in 250 mM imidazole, purified by gel filtration FPLC (Superdex-75; Amersham Pharmacia Biotech), and concentrated with an Amicon stirred ultrafiltration cell (Millipore). Proteins were stable for weeks at 4C in 50 mM Tris?HCl, pH 8.0/150 mM NaCl/1 mM PMSF/1 mM EDTA. Open in a separate windows Physique 1 Expression and purification of HD exon 1 constructs. (TRX (Promega). NMR Spectroscopy. 15N-labeled Methazathioprine HD-46Q or TRX-tag was expressed in minimal M9 medium prepared with 15N-labeled ammonium chloride (Aldrich) and purified. Samples were concentrated to 100 M in 50 mM sodium phosphate, pH 7.0/50 mM NaCl/5% D2O. Under these conditions, HD-46Q was not aggregated as shown by the elution of the post-NMR sample at the same time point as freshly prepared HD-46Q in gel filtration FPLC. Shigemi tubes were used to minimize the amount of Methazathioprine sample required. One-dimensional 1H and two-dimensional gradient-enhanced 1H,15N heteronuclear single quantum coherence (HSQC) NMR spectra were acquired at 4C on a Varian INOVA 600-MHz spectrometer equipped with a three-axis gradient HCN probe. A 2-sec relaxation delay and 8,610-Hz proton spectral width were used in all experiments. The one-dimensional spectra were acquired with 5,024 complex points and 2,048 transients; the solvent transmission was attenuated by presaturation. The two-dimensional spectra were acquired with 128 complex increments, 2,500-Hz nitrogen spectral width and 64 (TRX-tag) or 256 (HD-46Q) transients. Proton chemical shifts were referenced relative to TSP [3-(trimethylsilyl)propionic-2,2,3,3-repeating units (reddish dots). A monovalent ligand binds the lattice by interacting with several consecutive models. Binding of a first monovalent ligand occurs with an affinity that increases modestly with lattice length (e.g., 14 M for HD-25Q and 1.5 M for HD-46Q binding MW1 Fab, Table ?Table2).2). A second ligand molecule can bind to a poly(Gln) tract that contains more than one binding site, but binds with weaker affinity than the first ligand. A multivalent ligand can bind with high avidity to a longer poly(Gln) tract by interacting Methazathioprine with two binding Methazathioprine sites simultaneously (the macroscopic at concentrations below the because the ligand whose binding we have characterized (MW1 Fab) is derived.