The patch clamp technique for whole cell recordings was performed to specifically assess inhibitory functions of the different conantokins. would occur in Gla residues in an -helix at + 4, + 7, and + 11 in the absence of metal cations (22). As a result of this inclusion of Gla7, conG is usually unstructured in the absence of divalent ions, whereas conT, made up of Lys at position 7, exists as an -helix in the absence of divalent cations. This is likely due to favorable side chain charge-charge interactions with Lys7 and Gla residues 4 and 11 in helical turns (22, 23). Conantokins bearing systematic Ala substitutions at individual residues revealed the interdependence between the 5 and 6 N-terminal residues and NMDAR ion channel inhibitory properties (24). However, the relationships between the overall secondary structural characteristics of conantokins and main sequence-derived functional properties still remain unclear. Apart from the presence of Gla residues, some conotoxins contain 4-hydroxyproline (Hyp) (25), created post-translationally via prolyl-4-hydroxylase catalysis. In animals, this amino acid is found mainly in collagen/gelatin/elastin and stabilizes the collagen triple helix. Hyp also occurs in very small amounts in other proteins, cellular prion protein (26), argonaute 2 (27), hypoxia-inducible factor (28), and match C1q (29), with functional effects in secretion, protein folding, and stability, activity, receptor acknowledgement, and/or glycosylation. Additionally, conantokins from family that is closely related to the genus is usually of particular interest, because intuitively it would, at a minimum, negatively influence the ability of conantokins to form divalent cation-induced end-to-end helices. To determine the role of Hyp in the structure-function associations of conantokins, we combined SB 334867 a high resolution structural study of conRl-B, obtained by NMR spectroscopy, with evaluations of its ion channel antagonism of NMDARs in intact neurons made up of specific subunit gene inactivations. The results of these studies are summarized herein. Experimental Procedures Peptide Synthesis The following conantokins were chemically synthesized on a 0.1 mmol level using standard to 4-Hyp. axis pulsed field gradient cryoprobe, and running the TopSpin 3.2 pl6 software. The 1H, 13C, and 15N chemical shifts for each residue were determined by analyzing two-dimensional homonuclear DQF-COSY, TOCSY (60 ms), and NOESY (120 and 200 ms) spectra, as well as heteronuclear 1H-13C HSQC, HSQC-TOCSY, HMBC, and 1H-15N HSQC spectra. The spectra were measured using standard pulse sequences in the Bruker TopSpin 3.2, pl6 pulse sequence library. Suppression of the water transmission was accomplished by TET2 Watergate or Echo-Antiecho techniques. Proton chemical shifts were referenced to internal 4,4-dimethyl-4-silapentane-1-sulfonate. The 13C and 15N sizes in two-dimensional heteronuclear spectra were referenced indirectly (34). The NMR data were processed and analyzed with the programs TopSpin (Bruker Devices) and Sparky. Free induction decays were acquired with 2K complex data points for the TOCSY and NOESY data sets and with 4K complex data points for DQF-COSY experiments. A total of 400 + 2, and + 4) constraints were set to an upper SB 334867 bound of 6 ? and a lower bound of 1 1.8 ?. Dihedral angle restraints ( and ) were derived from chemical shift values of HN, N, CO, C, and C using TALOS+ (37, 38). The solution structures of conantokins were solved using standard simulated annealing protocols as implemented in Xplor-NIH 2.37 SB 334867 (39, 40). NOE-derived interproton distance restraints and dihedral angle restraints, predicted from NMR chemical shifts using TALOS+, were incorporated into restrained molecular dynamics followed by energy minimization with an all-atom pressure field. After global folding and refinement,.