Design of Cort-Ir-M1 variants.

<p>(A) Amino-acid sequence of the wild-type Cort-Ir coiled-coil dimer and its trimeric mutant Cort-Ir-M1. Heptad repeats (abcdefg) are indicated and shown as blocks. The experimentally determined trigger sequence is highlighted by a gray bar. Arginine and glutamate residues of the two complemented trimerization motifs I and II and additional trimer-specific isoleucine substitutions are highlighted in colour according to the amino acids’ physicochemical properties: blue, positively charged; red, negatively charged; green, hydrophobic, h, hydrophobic, x, any amino acid. (B) Amino- acid sequence of Cort-Ir-M1-short1 and Cort-Ir-M1-short2.</p

Prominent structural features seen in the Cort-Ir-M1-short2 coiled-coil trimer.

<p>(A) Side view of the salt-bridge networks formed between Arg7/14 and Glu12′/19′, and water-mediated hydrogen bond between Glu12′/19′:Oε2 and Arg7/14:O. The position of the two trimerization motifs I and II is indicated. (B) End-on view of the <b>a</b>2 layer showing the shielding of the Ile8 residues from solvent by the aliphatic side-chain moieties of Arg7. (C) End-on view of the <b>d</b>2 layer showing the hydrophobic packing between Ile11 and the aliphatic side-chain moieties of the Glu12 residues. Side chains of residues are shown in sticks representation and van der Waals spheres (B and D), the water molecules as small red spheres (A), and monomers A, B and C are shown as Cα-traces. Oxygen and nitrogen atoms are coloured in red and blue, and carbon atoms in cyan, yellow and grey for monomers A, B and C, respectively. The amino-acid sequence and sequence features of Cort-Ir-M1-short2 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063370#pone-0063370-g001" target="_blank">Figure 1</a>.</p

CD and sedimentation equilibrium analysis of Cort-Ir-M1-short1 and GFP-Cort-Ir-M1-short1.

<p>(A) Thermal unfolding profile of Cort-Ir-M1-short1 monitored by CD. The CD signal at 222 nm was followed by increasing the temperature at a rate of 1°C/min. The midpoint of unfolding, T_m, is centred at 69°C at a protein concentration of 13 µM. Inset, CD spectrum of Cort-Ir-M1-short1 at 5°C. (B) Analysis of the oligomerization state of GFP-Cort-Ir-M1-short1 by sedimentation-equilibrium analysis. The protein was analyzed at concentrations of 55, 110 and 220 µM in PBS. (C) Analysis of the oligomerization state of Cort-Ir-M1-short by sedimentation-equilibrium analysis. The protein was analyzed at a concentration of 5 µM in PBS. The data were collected at 235 nm. All AUC data were globally fitted according to a single ideal species model and in all cases the derived masses are consistent with the presence of trimmers only. Concentrations refer to monomer equivalents.</p

Data collection and refinement statistics.

a<p>Values in parentheses correspond to the highest resolution shell.</p>b<p>As defined by Karplus & Diederichs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063370#pone.0063370-Karplus1" target="_blank">[34]</a>.</p>c<p>Statistics from Molprobity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063370#pone.0063370-Chen2" target="_blank">[35]</a>.</p

Sedimentation velocity analysis of GFP-Cort-Ir-M1-short1.

<p>Fluorescence-monitored AUC sedimentation velocity of GFP-Cort-Ir-M1-short1. The protein concentrations were 5, 50 and 500 nM (monomer equivalents). The fluorescence of GFP was monitored at 488 nm. Traces and residuals are shown for the experiment carried out at a protein concentration of 50 nM. The distribution of sedimentation coefficients indicates the presence of only monomeric and trimeric species only. The fitted masses for single ideal species are listed in the lower panel. The dissociation constant, K_D, of GFP-Cort-Ir-M1-short was estimated to be 10⁻¹⁴ M⁻².</p