Synthesis of α-hydroxy threonine (Tah, 2R, 3S-dihydroxy-butanoic acid) (1). L-Threonine (2.2 g, 18.5 mmol), suspended in 5 ml of water at -5 °C, was treated simultaneously with a solution of 1.38 g NaNO 2 (20 mmol) in 2 ml of water and 557 µl of concentrated H 2 SO 4 (10 mmol) in 1.5 ml H 2 O. The two solutions were added slowly while stirring so that the temperature remained between 0 °C and 5 °C. The reaction turned yellow upon addition. The solution was then stirred overnight at room temperature. The reaction mixture was concentrated, the mixture treated with 3 ml of EtOH, and the salts were filtered. The solution was concentrated. The material was dry loaded onto a flash silica gel column and run in 1:1 hexanes/ethyl acetate with 1% acetic acid to give 730 mg (38 %) of hydroxythreonine Synthesis of Tah cyanomethyl ester (2R, 3S-dihydroxy-butanoate cyanomethyl ester) (2).The hydroxy acid (385 mg, 3.21 mmol) was dissolved in 5.1 ml of ClCH 2 CN (80.1 mmol) and 1.2 ml Et 3 N (8.44 mmol). Upon stirring under Ar for 30 min, the solution turned yellow. A gradient flash silica gel column from 20% to 80% ethyl acetate/hexanes was run, and the isolated product was dried on vacuum to yield 50.9 mg (10%) of hydroxythreonine cyanomethyl ester: 1 H NMR (D 2 O) δ 1.27 (d, 3H, J= 6 Hz), 4.22 (m, 1H), 4.34 (d, 1H, J= 3 Hz), 5.01 (s,

The nicotinic acetylcholine receptor and related Cys-loop receptors are ligand-gated ion channels that mediate fast synaptic transmission throughout the central and peripheral nervous system. A highly conserved aspartate residue (D89) that is near the agonist binding site, but does not directly contact the ligand, plays a critical part in receptor function. Here we probe the role of D89 using unnatural amino acid mutagenesis coupled with electrophysiology. Homology modeling implicates several hydrogen bonds involving D89. We find that no single hydrogen bond is essential to proper receptor function. Apparently the side chain of D89 establishes a redundant network of hydrogen bonds; these bonds preorganize the agonist binding site by positioning a critical tryptophan residue that directly contacts the ligand. Earlier studies of the D89N mutant led to the proposal that a negative charge at this position is essential for receptor function. However, we find that receptors with neutral side chains at position 89 can function well, if the side chain is less perturbing than the amide of asparagine (nitro or keto groups allow function), or if a compensating backbone mutation is made to relieve unfavorable electrostatics.Neuroreceptors are central players of synaptic transmission, receiving and interpreting chemical signals between neurons in the nervous system. Neuroreceptors of the ligand-gated ion channel (LGIC) family convert incoming chemical signals into electrical output. In the LGIC gating process, neurotransmitters are recognized by ligand-binding domains, and binding triggers conformational changes within the structure to form an ion-conducting pore.The nicotinic acetylcholine receptor (nAChR) has served as a prototype for understanding the structure and function of the Cys-loop family of LGICs (also known as pentameric LGICs). In this superfamily of receptors, which also includes γ-aminobutyric acid, glycine, and serotonin receptors, the five subunits are symmetrically or pseudosymmetrically arranged around a central ion-conducting pore. Each subunit contains a four-helix transmembrane domain that contains the ion channel gate and an extracellular ligandbinding domain. Members of nAChR family are expressed at the neuromuscular junction and in the electric organ of eels and rays (muscle-type receptors), as well as in the central nervous system (neuronal receptors). The muscle-type receptor is the best characterized, and the form studied here is the embryonic muscle nAChR, with a subunit stoichiometry of two α1 subunits and one each of β1, γ, and δ subunits(1). The nAChR has two agonist binding sites located at the α/γ and α/δ subunit interfaces(2-4). The α subunits contribute the primary * Author to whom correspondence should be addressed. Phone (626) Fax (626) 564-9297; dadougherty@caltech.edu. † These authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 September 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-...