Ca²⁺ binding to VILIP-3.

<p>(A) Ca²⁺ binding ITC isotherm for myristoylated VILIP-3. The overall Ca²⁺ binding stoichiometry is 3:1, consistent with Ca²⁺ bound at EF2, EF3 and EF4. The average dissociation constant (K_d) and binding enthalpy (ΔH) for the three sites are 0.3 μM and –6.4 kcal/mol, respectively. (B) Ca²⁺-binding data measured by flow dialysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165921#pone.0165921.ref033" target="_blank">33</a>]. Representative Ca²⁺ binding data for VILIP-3 wildtype (black squares) and Y64A (open circles) are shown. Fitted curves (dashed lines) were calculated using the Hill model. Fitting parameters for wild type and mutants are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165921#pone.0165921.t001" target="_blank">Table 1</a>.</p

Structural Statistics for Ca²⁺-free VILIP-3.

<p>Structural Statistics for Ca²⁺-free VILIP-3.</p

NMR spectroscopy of myristoylated VILIP-3.

<p>Two-dimensional (¹H-¹⁵N HSQC) NMR spectra are shown for ¹⁵N-labeled VILIP-3 in the Ca²⁺-free (A) and Ca²⁺-bound (B) states. Spectra were recorded at 600 MHz at 30°C.</p

NMR spectroscopy of myristoyl binding site in VILIP-3.

<p>Three-dimensional ¹³C(F1)-edited ¹³C(F3)-filtered HMQC NOESY spectra of unlabeled VILIP-3 containing a ¹³C-labeled myristoyl group. (A-C) Two-dimensional slices of (¹³C/F1)-edited (¹³C/F3)-filtered HMQC NOESY spectra of ¹³C-labeled myristoyl group attached to Ca²⁺-free myristoylated VILIP-3 edited at ¹³C frequencies 16.89 (A), 34.31 (B) and 37.94 (C) ppm. NOE crosspeaks represent protons of myristate that are less than 5 Å away from aliphatic and aromatic resonances of the Ca²⁺-free protein (marked by residue labels). Schematic view of protein contacts along fatty acyl chain (D) and same view rotated by 180° (E). Side-chain atoms of hydrophobic residues near fatty acyl chain are highlighted yellow.</p

NMR-derived structures of myristoylated VILIP-3 (PDB ID: 5T7C).

<p>Ribbon diagrams show an overlay of the 10 lowest energy structures (A), and average main chain structure of Ca²⁺-free VILIP-3 (B) and rotated by 180° (C) with N-terminal myristoyl group highlighted magenta. The unstructured N-terminal loop region is depicted by a dashed line. Secondary structure elements (helices and strands) and EF-hand motifs are drawn in the same colors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165921#pone.0165921.g001" target="_blank">Fig 1</a> (EF1 green, EF2 red, EF3 cyan and EF4 yellow). Side-chain atoms of hydrophobic residues at the domain interface (A) and that contact the myristoyl group (B) are highlighted yellow.</p

Amino acid sequence alignment of human VILIP-3 with other NCS proteins

<p>Secondary structure elements (helices and strands derived from NMR chemical shifts) and EF-hand motifs (EF1 green, EF2 red, EF3 cyan and EF4 yellow) are shown above the amino acid sequence of VILIP-3. Residues in VILIP-3 that interact with the myristoyl group are highlighted in bold and blue. Swiss Protein Database accession numbers are P37235 (human VILIP-3), P21457 (bovine recoverin), P62166 (human NCS-1), P46065 (bovine GCAP1).</p

¹H and ¹³C (in parentheses) chemical shift assignments of myristoyl resonances.

<p>¹H and ¹³C (in parentheses) chemical shift assignments of myristoyl resonances.</p

Folding stability of VILIP-3.

<p>Representative DSC thermograms for Ca²⁺-free VILIP-3 in both unmyristoylated (black) and myristoylated (green) states. The protein unfolding temperatures (T_M) determined from the thermograms are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165921#pone.0165921.t001" target="_blank">Table 1</a>. The T_M for Ca²⁺-saturated myristoylated VILIP-3 could not be accurately measured due to complications caused by Ca²⁺-induced protein aggregation (see blue trace).</p

Ca²⁺ Binding and Folding Stability of VILIP-3, Recoverin and Mutants.

<p>Ca²⁺ Binding and Folding Stability of VILIP-3, Recoverin and Mutants.</p

Conformational exchange of CfAFP-501 observed at 17°C (A) but not at −3°C (B).

<p>The difference of apparent R₂ at τ_cp value of 1.0 and 10.0 ms, i.e., ΔR₂(τ_cp)  =  R₂(10.0 ms)- R₂(1.0 ms), is plotted versus residue number.</p