NMR Structure of Retinal Guanylate Cyclase Activating Protein 5 (GCAP5) with R22A Mutation That Abolishes Dimerization and Enhances Cyclase Activation.

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5R22A) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5WT). Here, we present ITC, NMR, and functional analysis of GCAP5R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5R22A binds a total of 3 Ca2+, including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5WT were not detected by ITC, suggesting that R22A may affect the binding of Ca2+ to these sites. The NMR-derived structure of GCAP5R22A is overall similar to that of GCAP5WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5R22A with ∼25% higher affinity compared to GCAP5WT, consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Chemical shift assignments of retinal guanylyl cyclase activating protein 5 (GCAP5) with a mutation (R22A) that abolishes dimerization and enhances cyclase activation.

Retinal membrane guanylyl cyclases (RetGCs) in vertebrate rod and cone photoreceptors are activated by a family of neuronal Ca2+ sensor proteins called guanylyl cyclase activating proteins (GCAP1-7). GCAP5 from zebrafish photoreceptors binds to RetGC and confers Ca2+/Fe2+-dependent regulation of RetGC enzymatic activity that promotes the recovery phase of visual phototransduction. We report NMR chemical shift assignments of GCAP5 with a R22A mutation (called GCAP5R22A) that abolishes protein dimerization and activates RetGC with 3-fold higher activity than that of wild type GCAP5 (BMRB No. 51,783).

DNA Photocleavage in the Near-Infrared Wavelength Range by 2-Quinolinium Dicarbocyanine Dyes.

Here, we report the syntheses of two pentamethine cyanine dyes containing quinolinium rings and substituted with either hydrogen (3) or bromine (4) at the meso carbon. The electron withdrawing bromine atom stabilizes dye 4 in aqueous buffer, allowing complex formation to occur between the dye and double-helical DNA. UV-visible, CD, and fluorescence spectra recorded at low DNA concentrations suggest that dye 4 initially binds to the DNA as a high-order aggregate. As the ratio of DNA to dye is increased, the aggregate is converted to monomeric and other low-order dye forms that interact with DNA in a non-intercalative fashion. The brominated dye 4 is relatively unreactive in the dark, but, under 707-759 nm illumination, generates hydroxyl radicals that cleave DNA in high yield (pH 7.0, 22 °C). Dye 4 is also taken up by ES2 ovarian carcinoma cells, where it is non-toxic under dark conditions. Upon irradiation of the ES2 cells at 694 nm, the brominated cyanine reduces cell viability from 100 ± 10% to 14 ± 1%. Our results suggest that 2-quinolinium-based carbocyanine dyes equipped with stabilizing electron withdrawing groups may have the potential to serve as sensitizing agents in long-wavelength phototherapeutic applications.