Fig. 3.

Hypothetical tandem linker conformations. Several linker architectures reported in the literature for GABA_ARs were evaluated for linker strain using molecular mechanics models. Here, we present the optimized tandem-containing receptors in each possible tandem configuration: counterclockwise (left), clockwise (center), and loop-over (right). The color code for subunits is α₁ (teal), β₂ (gray), and γ₂ (blue). Linkers are hot pink, and regions of the native subunit allowed flexibility are shown in purple. Evaluated linkers were β-23-α (A–C) with linker composed of Q₃(Q₂A₃PA)₂AQ₅, β-10Q-α (D–F), α-10Q-β (G–I), all tested in oocytes by Baumann and coworkers (9). Also tested but not shown were α-(Q9+signal peptide)-β (11,12), β-(10Q+signal peptide)-α from Fig. 2, and γ-26-β (20). The latter three showed no energetic differences in linker strain for counterclockwise, clockwise, or loop-over conformations. To see differences in relative strain among the tandem arrangements for each linker, ΔE was calculated by subtracting the converged potential energy of linker-imposed strain from the energy for the counterclockwise conformation. Data are mean ± SD from three or more converged cycles. For these calculations, all parts of the pentamer were held fixed except for linker sequence and adjoining terminal regions (3-31 residues, bottom). Linker and signal peptides were added to our core model and assigned Gasteiger-Marsili charges and Sybyl atom types from the Tripos forcefield by default. Each structure was minimized 4000 steps by default methods and was then subjected to 8 to 15 2-picosecond cycles of simulated annealing in vacuo with a linear temperature gradient from 700 to 200 K, followed by a quenching minimization after each cycle. Cycles were repeated until apparent convergence of system potential energy.