Amino acid sequence alignment of GCAP1 with various NCS proteins.

<p>Secondary structural elements are indicated schematically. The four EF-hands (EF1, EF2, EF3 and EF4) are underlined. Residues mutated in EF4mut (D144N/D148G) are indicated in red. Residues at the domain interface (V77 – L97) that have broadened NMR resonances are shown in italics. </p

Schematic model of conformational changes in GCAP1 caused by Ca²⁺-binding at EF4.

<p>(A) Structural model of EF4mut activator state was generated by homology modeling using the NMR structure of recoverin (1jsa) that contains Ca²⁺ bound at EF2 and EF3 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081822#B23" target="_blank">23</a>]. The four EF-hands are colored green (EF1), red (EF2), cyan (EF3) and yellow (EF4), and bound Ca²⁺ is orange. (B) The crystal structure of Ca²⁺-saturated GCAP1 (2r2i) shows key hydrophobic residues at the domain interface are solvent exposed in the Ca²⁺-bound inhibitor state. The Ca²⁺-dependent rearrangement of the W94 side-chain at the domain interface might control the switching between activator and inhibitor states. The N-terminal myristoyl group (magenta) is sequestered inside the protein in both structures.</p

Structural characterization of GCAP1 mutants at the domain interface.

<p>(A) Overlay of ¹⁵N-¹H HSQC spectra of V77E (red), L82E (blue), WT (black) and W94F (green) indicate that each mutant is properly folded and structurally intact. The mutant spectra look similar to that of wildtype particularly for residues in structured regions. Minor spectral differences are observed for exposed residues in unstructured regions, most likely due to small differences in solvent conditions. (B) Expanded view of ¹⁵N-¹H HSQC downfield region for GCAP1 mutants: V77E (red), L82E (blue), WT (black) and W94F (green). Three downfield NMR peaks assigned to G69, G105 and G149 indicate Ca²⁺ is bound functionally at EF2, EF3 and EF4 for each mutant.</p

NMR chemical shift mapping for GCAP1.

<p>(A) Amide chemical shift differences between EF4mut and Ca²⁺-saturated wildtype (CSD = {(H_N^A – H_N^I)² + (¹⁵N^A – ¹⁵N^I)²}^1/2 , where “A” and “I” designate activator and inhibitor states) plotted as a function of residue number. (B) CSD values from part “A” are mapped onto the GCAP1 crystal structure (2R2I). Residues with the largest chemical shift difference (CSD > 0.12 ppm) are highlighted in red, intermediate chemical shift differences (0.06 < CSD < 0.12) shown in yellow, and smallest chemical shift differences (CSD < 0.06) shown in green. Unassigned residues are colored gray and the myristoyl group is orange.</p

NMR spectra of activator vs. inhibitor forms of GCAP1.

<p>Two-dimensional (¹H-¹⁵N HSQC) NMR spectra of ¹⁵N-labeled wildtype Ca²⁺-saturated GCAP1 (A) and EF4mut (B). Spectra were obtained at 37 °C in the presence of 40 mM octylglucoside. Downfield resonances (~10.5 ppm) are assigned to conserved glycine residues in each Ca²⁺-bound EF-hand loop. For Ca²⁺-saturated GCAP1 (A), downfield peaks assigned to G69, G105 and G149 indicate Ca²⁺ is bound at EF2, EF3 and EF4. For EF4mut (B), downfield peaks assigned to G69 and G105 indicate Ca²⁺ is bound at EF2 and EF3 in EF4mut. Sequence specific resonance assignments for Ca²⁺-saturated GCAP1 were determined previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081822#B31" target="_blank">31</a>].</p

Changes in flash responses after deletion of GCAP1.

<p>Averaged flash responses of a WT rod (<b>A</b>) peaked sooner and had a reduced tail component in the recovery compared to two GCAP1^−/−rods designated arbitrarily as having “fast” (<b>B</b>) or “slow” (<b>C</b>) response kinetics (marked accordingly as “fast” and “slow” in the panels). Maximal response amplitudes were 11, 10 and 14 pA, respectively. The integration times of dim flash responses, whose amplitudes were less than 20% of the maximal response, were 250 ms for the WT rod and 236 and 483 ms for the two GCAP1^−/− rods. The flash was presented at time zero. Flash strengths were: 14, 31, 58, 121, 227, 505, 945, 1973 and 3691 photons µm⁻² for the WT rod; 6, 11, 23, 44, 91, 171, 380, 713, 1482, 2773 and 6091 photons µm⁻² for the GCAP1^−/− rod in <b>B</b> and 3, 5, 20, 80, 311, 692, 1300, 2691 and 5045 photons µm⁻² for the GCAP1^−/− rod in <b>C.</b> Records were digitally filtered at 12 Hz. <b>D.</b> Stepped recovery of the bright flash response in two trials for the WT rod in <b>A</b> due to aberrant photon responses. Flash strength was 3691 photons µm⁻². The number of steps and the temporal depth of their tread varied randomly from trial to trial. <b>E.</b> Tendency for steps to be larger in GCAP1^−/− rods. Responses were recorded from a GCAP1^−/− rod different from those in <b>B</b> and <b>C.</b> Flash strength was 2773 photons µm⁻². Records were digitally filtered at 8 Hz. <b>F.</b> Average stimulus-response relations for 28 WT (<i>black</i>) and 36 GCAP1^−/− (<i>red</i>) rods. Each circle averages the normalized responses of several rods that were grouped by similar flash strength, and error bars show SEM. Continuous lines show the saturating exponential function <i>r/r_max = 1− exp(-ki)</i>, where <i>i</i> is flash strength, <i>k</i> is equal to ln(2)/i_0.5, and <i>i_0.5</i> is the flash strength that produces a half-saturating response, with i_0.5 values of 66 and 23 photons µm⁻² for WT and GCAP1^−/−, respectively. These i_0.5 values were derived from the mean <i>k</i> from fits to individual WT and GCAP1^−/− rods. <b>G.</b> Stimulus-response relations for the tail of saturated responses from 16 WT (<i>black</i>) and 35 GCAP1^−/− (<i>red</i>) rods, measured at 1.5 (<i>thick symbols</i>) and 2 s (<i>thin symbols</i>) after the flash. Each symbol represents the average, normalized response amplitude of 12 to 15 WT rods or 24 to 30 KO rods (except at the lowest and highest flash strengths, for which there were only 1–6 rods), where groups were made according to flash strength. Error bars for flash strength are shown although variation was negligible on a log scale. Continuous lines show saturating exponential functions with averaged values for <i>k</i> (see above) derived from fits to individual rods. <b>H.</b> Pepperberg plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#pone.0047637-Pepperberg1" target="_blank">[34]</a> for 11 WT (<i>black</i>) and 28 GCAP1^−/− (<i>red</i>) rods. The saturation time of a bright flash response was measured from mid-flash to the point at which the saturation response declined to 0.8 r_max, i.e., 20% recovery, as demarcated by the dotted lines in <b>A–C</b>. Results from each rod were plotted with a different symbol. The continuous lines have slopes equal to τ_c of 191 ms for WT and 159 ms for GCAP1^−/−, that were the mean values of linear regressions from individual rods in each group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#pone-0047637-t002" target="_blank"><b>Table 2</b></a>).</p

Photoreceptor protein expression in GCAP1^−/− retina.

<p><b>A.</b> Immunoblots of SDS polyacrylamide gels containing samples from WT and GCAP1 KO retinas probed with antibodies raised against GCAP2, RetGC1, RetGC2, rod α-transducin (Gtα1), PDE6, arrestin 1 (ARR), GRK1, RGS9, and β-actin, as indicated. <b>B.</b> Average (± SD) integrated chemiluminescence signal intensity in the band for the corresponding antigen in GCAP1^−/− retina relative to the WT for GCAP1 (n = 5), GCAP2 (n = 7), RetGC1 (n = 3), RetGC2 (n = 3), rod α-transducin (n = 3), PDE6 (n = 3), arrestin (n = 3), GRK1 (n = 3), RGS9 (n = 3), and β-actin (n = 3). When compared by one-way ANOVA with Bonferroni’s <i>post hoc</i> test (alpha = 0.01), there were significant differences found in GCAP1 (**) and GCAP2 (*) contents (P<0.0001 and P<0.006, respectively), but not in other tested proteins.</p

Recovery of bright flash response of rods, reconstructed from recordings of paired-flash ERGs.

<p><b>A.</b> Fractional a-wave recovery from a strong flash, presented at time zero, in paired-flash ERGs from 16 WT (•) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#pone.0047637-Olshevskaya1" target="_blank">[22]</a>, 17 GCAPs1,2^−/− (▵), and 17 GCAP1^−/− (○) mice aged 1.5–3 months. <b>B.</b> The recovery remained fast in the absence of each RetGC isozyme; 16 WT (•), 17 GCAP1^−/− (○), 18 RetGC1^−/−GCAP1^−/− (⋄), and 17 RetGC2^−/−GCAP1^−/− (□) mice. The average saturating a-wave amplitudes in WT, GCAP1^−/−, GCAPs1,2^−/−, RetGC1^−/−GCAP1^−/−, and RetGC2^−/−GCAP1^−/− were 532, 347, 365, 98, and 277 µV, respectively. The continuous curves were ‘smooth line’ fit by KaleidaGraph software. In many cases, only the initial phase of the ERG recovery could be fit by a single exponential. The time required for 50% amplitude recovery determined from the exponential portion of the fit in 16 mice for each genotype was (mean ± SEM): 0.55±0.02, 0.51±0.02, 0.50±0.02, 0.51±0.02, and 1.78±0.06 s in WT, GCAP1^−/−, RetGC1^−/−GCAP1^−/−, RetGC2^−/−GCAP1^−/−, and GCAPs12^−/−, respectively. In all-pairs comparison, the only significant difference for the entire group (P<0.0001, one way ANOVA with a Bonferroni post-hoc test, alpha = 0.01) was found between GCAPs1,2^−/− and all other genotypes. Contribution of a small fraction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#pone.0047637-Jeon1" target="_blank">[45]</a> of mouse cones to the scotopic a-wave amplitude was considered insignificant for this analysis.</p

Rod outer segment morphology.

<p>Measurements were made on rods from the central retina of 2 or 3 mice of each type, aged 2–3 months (representative sections are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#pone-0047637-g002" target="_blank"><b>Fig. 2F, G</b></a>). Data are given as mean ± SEM, (number of rods measured, P-value from a t-test for values less than 0.05).</p

Altered RetGC activity in GCAP1^−/− mouse retinas.

<p>Total (<b>A)</b> and normalized (<b>B</b>) cGMP synthetic activity in WT (•, n = 5) and GCAP1^−/− (○, n = 4) retinas as a function of free Ca²⁺ concentration. Notice that sensitivity shifted to lower levels of Ca²⁺ in GCAP1^−/− retinas. In panel <b>B</b>, the activities in each series were normalized by the corresponding maximal RetGC activity measured in each genotype and averaged for each group. The data were fitted by the equation, <i>A = (A_max – A_min)/(1+([Ca]/[Ca]_1/2)^h) + A_min</i>; where <i>A_max</i> and <i>A_min</i> are the maximal and the minimal activity of guanylyl cyclase, respectively, <i>[Ca]_1/2</i> is the concentration of Ca²⁺ producing 50% inhibition, and <i>h</i> is a Hill coefficient. RetGC activity was assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047637#s2" target="_blank">Materials and Methods</a>. A_max values for the WT and GCAP1^−/− retinas were 0.6 and 0.8 nmol cGMP min⁻¹ retina⁻¹, [Ca]_1/2 values were 81 and 46 nM, and <i>h</i> values were 1.8 and 1.6, respectively.</p