Correlating structural and photochemical heterogeneity in cyanobacteriochrome NpR6012g4.

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.

Retinal degeneration 3 (RD3) protein, a retinal guanylyl cyclase regulator, forms a monomeric and elongated four-helix bundle.

Retinal degeneration 3 (RD3) protein promotes accumulation of retinal membrane guanylyl cyclase (RetGC) in the photoreceptor outer segment and suppresses RetGC activation by guanylyl cyclase-activating proteins (GCAPs). Mutations truncating RD3 cause severe congenital blindness by preventing the inhibitory binding of RD3 to the cyclase. The high propensity of RD3 to aggregate in solution has prevented structural analysis. Here, we produced a highly soluble variant of human RD3 (residues 18-160) that is monomeric and can still bind and negatively regulate RetGC. The NMR solution structure of RD3 revealed an elongated backbone structure (70 Å long and 30 Å wide) consisting of a four-helix bundle with a long unstructured loop between helices 1 and 2. The structure reveals that RD3 residues previously implicated in the RetGC binding map to a localized and contiguous area on the structure, involving a loop between helices 2 and 3 and adjacent parts of helices 3 and 4. The NMR structure of RD3 was validated by mutagenesis. Introducing Trp85 or Phe29 to replace Cys or Leu, respectively, disrupts packing in the hydrophobic core and lowers RD3's apparent affinity for RetGC1. Introducing a positive charge at the interface (Glu32 to Lys) also lowered the affinity. Conversely, introducing Val in place of Cys93 stabilized the hydrophobic core and increased the RD3 affinity for the cyclase. The NMR structure of RD3 presented here provides a structural basis for elucidating RD3-RetGC interactions relevant for normal vision or blindness.

Structural Characterization of Ferrous Ion Binding to Retinal Guanylate Cyclase Activator Protein 5 from Zebrafish Photoreceptors.

Sensory guanylate cyclases (zGCs) in zebrafish photoreceptors are regulated by a family of guanylate cyclase activator proteins (called GCAP1-7). GCAP5 contains two nonconserved cysteine residues (Cys15 and Cys17) that could in principle bind to biologically active transition state metal ions (Zn2+ and Fe2+). Here, we present nuclear magnetic resonance (NMR) and isothermal titration calorimetry (ITC) binding analyses that demonstrate the binding of one Fe2+ ion to two GCAP5 molecules (in a 1:2 complex) with a dissociation constant in the nanomolar range. At least one other Fe2+ binds to GCAP5 with micromolar affinity that likely represents electrostatic Fe2+ binding to the EF-hand loops. The GCAP5 double mutant (C15A/C17A) lacks nanomolar binding to Fe2+, suggesting that Fe2+ at this site is ligated directly by thiolate groups of Cys15 and Cys17. Size exclusion chromatography analysis indicates that GCAP5 forms a dimer in the Fe2+-free and Fe2+-bound states. NMR structural analysis and molecular docking studies suggest that a single Fe2+ ion is chelated by thiol side chains from Cys15 and Cys17 in the GCAP5 dimer, forming an [Fe(SCys)4] complex like that observed previously in two-iron superoxide reductases. Binding of Fe2+ to GCAP5 weakens its ability to activate photoreceptor human GC-E by decreasing GC activity >10-fold. Our results indicate a strong Fe2+-induced inhibition of GC by GCAP5 and suggest that GCAP5 may serve as a redox sensor in visual phototransduction.

Structure of Guanylyl Cyclase Activator Protein 1 (GCAP1) Mutant V77E in a Ca2+-free/Mg2+-bound Activator State.

GCAP1, a member of the neuronal calcium sensor subclass of the calmodulin superfamily, confers Ca(2+)-sensitive activation of retinal guanylyl cyclase 1 (RetGC1). We present NMR resonance assignments, residual dipolar coupling data, functional analysis, and a structural model of GCAP1 mutant (GCAP1(V77E)) in the Ca(2+)-free/Mg(2+)-bound state. NMR chemical shifts and residual dipolar coupling data reveal Ca(2+)-dependent differences for residues 170-174. An NMR-derived model of GCAP1(V77E) contains Mg(2+) bound at EF2 and looks similar to Ca(2+) saturated GCAP1 (root mean square deviations = 2.0 Å). Ca(2+)-dependent structural differences occur in the fourth EF-hand (EF4) and adjacent helical region (residues 164-174 called the Ca(2+) switch helix). Ca(2+)-induced shortening of the Ca(2+) switch helix changes solvent accessibility of Thr-171 and Leu-174 that affects the domain interface. Although the Ca(2+) switch helix is not part of the RetGC1 binding site, insertion of an extra Gly residue between Ser-173 and Leu-174 as well as deletion of Arg-172, Ser-173, or Leu-174 all caused a decrease in Ca(2+) binding affinity and abolished RetGC1 activation. We conclude that Ca(2+)-dependent conformational changes in the Ca(2+) switch helix are important for activating RetGC1 and provide further support for a Ca(2+)-myristoyl tug mechanism.

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Protonated Bilin Ring System in Both Photostates.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. Both CBCRs and phytochromes use photoisomerization of a linear tetrapyrrole (bilin) chromophore to photoconvert between two states with distinct spectral and biochemical properties, the dark state and the photoproduct. The isolated CBCR domain NpR6012g4 from Nostoc punctiforme is a well-characterized member of the canonical red/green CBCR subfamily, photosensory domains that can function as sensors for light color or intensity to regulate phototactic responses of filamentous cyanobacteria. Such red/green CBCRs utilize conserved Phe residues to tune the photoproduct for green light absorption, but conflicting interpretations of the photoproduct chromophore structure have been proposed. In the hydration model, the proposed photoproduct state is extensively solvated, with a loosely bound, conformationally flexible chromophore. In the trapped-twist model, the photoproduct chromophore is sterically constrained by hydrophobic amino acids, including the known Phe residues. Here, we have characterized chromophore structure in NpR6012g4 using solution nuclear magnetic resonance spectroscopy and a series of labeled chromophores. Four NH resonances are assigned for both the red-absorbing dark state and the green-absorbing photoproduct. Moreover, observed (13)C chemical shifts are in good agreement with those obtained for protonated rather than deprotonated bilins in ab initio calculations. Our results demonstrate that NpR6012g4 has a protonated, cationic bilin π system in both photostates, consistent with a photoproduct structure in which the chromophore is not extensively hydrated.

Characterization of Red/Green Cyanobacteriochrome NpR6012g4 by Solution Nuclear Magnetic Resonance Spectroscopy: A Hydrophobic Pocket for the C15-E,anti Chromophore in the Photoproduct.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins distantly related to phytochromes. Like phytochromes, CBCRs reversibly photoconvert between a dark-stable state and a photoproduct via photoisomerization of the 15,16-double bond of their linear tetrapyrrole (bilin) chromophores. CBCRs provide cyanobacteria with complete coverage of the visible spectrum and near-ultraviolet region. One CBCR subfamily, the canonical red/green CBCRs typified by AnPixJg2 and NpR6012g4, can function as sensors of light color or intensity because of their great variation in photoproduct stability. The mechanistic basis for detection of green light by the photoproduct state in this subfamily has proven to be a challenging research topic, with competing hydration and trapped-twist models proposed. Here, we use ¹³C-edited and ¹5N-edited ¹H-¹H NOESY solution nuclear magnetic resonance spectroscopy to probe changes in chromophore configuration and protein-chromophore interactions in the NpR6012g4 photocycle. Our results confirm a C15-Z,anti configuration for the red-absorbing dark state and reveal a C15-E,anti configuration for the green-absorbing photoproduct. The photoactive chromophore D-ring is located in a hydrophobic environment in the photoproduct, surrounded by both aliphatic and aromatic residues. Characterization of variant proteins demonstrates that no aliphatic residue is essential for photoproduct tuning. Taken together, our results support the trapped-twist model over the hydration model for the red/green photocycle of NpR6012g4.

Identification of target binding site in photoreceptor guanylyl cyclase-activating protein 1 (GCAP1).

Retinal guanylyl cyclase (RetGC)-activating proteins (GCAPs) regulate visual photoresponse and trigger congenital retinal diseases in humans, but GCAP interaction with its target enzyme remains obscure. We mapped GCAP1 residues comprising the RetGC1 binding site by mutagenizing the entire surface of GCAP1 and testing the ability of each mutant to bind RetGC1 in a cell-based assay and to activate it in vitro. Mutations that most strongly affected the activation of RetGC1 localized to a distinct patch formed by the surface of non-metal-binding EF-hand 1, the loop and the exiting helix of EF-hand 2, and the entering helix of EF-hand 3. Mutations in the binding patch completely blocked activation of the cyclase without affecting Ca(2+) binding stoichiometry of GCAP1 or its tertiary fold. Exposed residues in the C-terminal portion of GCAP1, including EF-hand 4 and the helix connecting it with the N-terminal lobe of GCAP1, are not critical for activation of the cyclase. GCAP1 mutants that failed to activate RetGC1 in vitro were GFP-tagged and co-expressed in HEK293 cells with mOrange-tagged RetGC1 to test their direct binding in cyto. Most of the GCAP1 mutations introduced into the "binding patch" prevented co-localization with RetGC1, except for Met-26, Lys-85, and Trp-94. With these residues mutated, GCAP1 completely failed to stimulate cyclase activity but still bound RetGC1 and competed with the wild type GCAP1. Thus, RetGC1 activation by GCAP1 involves establishing a tight complex through the binding patch with an additional activation step involving Met-26, Lys-85, and Trp-94.

Photoconversion changes bilin chromophore conjugation and protein secondary structure in the violet/orange cyanobacteriochrome NpF2164g3' [corrected].

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors distantly related to phytochromes. All CBCRs examined to date utilize a conserved Cys residue to form a covalent thioether linkage to the bilin chromophore. In the insert-Cys CBCR subfamily, a second conserved Cys can covalently link to the bilin C10 methine bridge, allowing detection of near-UV to blue light. The best understood insert-Cys CBCR is the violet/orange CBCR NpF2164g3 from Nostoc punctiforme, which has a stable second linkage in the violet-absorbing dark state. Photoconversion of NpF2164g3 leads to elimination of the second linkage and formation of an orange-absorbing photoproduct. We recently reported NMR chemical shift assignments for the orange-absorbing photoproduct state of NpF2164g3. We here present equivalent information for its violet-absorbing dark state. In both photostates, NpF2164g3 is monomeric in solution and regions containing the two conserved Cys residues essential for photoconversion are structurally disordered. In contrast to blue light receptors such as phototropin, NpF2164g3 is less structurally ordered in the dark state than in the photoproduct. The insert-Cys insertion loop and C-terminal helix exhibit light-dependent structural changes. Moreover, a motif containing an Asp residue also found in other CBCRs and in phytochromes adopts a random-coil structure in the dark state but a stable α-helix structure in the photoproduct. NMR analysis of the chromophore is consistent with a less ordered dark state, with A-ring resonances only resolved in the photoproduct. The C10 atom of the bilin chromophore exhibits a drastic change in chemical shift upon photoconversion, changing from 34.5 ppm (methylene) in the dark state to 115 ppm (methine) in the light-activated state. Our results provide structural insight into the two-Cys photocycle of NpF2164g3 and the structurally diverse mechanisms used for light perception by the larger phytochrome superfamily.

Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity.

Guanylyl cyclase-activating protein 1 (GCAP1), a myristoylated Ca(2+) sensor in vision, regulates retinal guanylyl cyclase (RetGC). We show that protein-myristoyl group interactions control Ca(2+) sensitivity, apparent affinity for RetGC, and maximal level of cyclase activation. Mutating residues near the myristoyl moiety affected the affinity of Ca(2+) binding to EF-hand 4. Inserting Phe residues in the cavity around the myristoyl group increased both the affinity of GCAP1 for RetGC and maximal activation of the cyclase. NMR spectra show that the myristoyl group in the L80F/L176F/V180F mutant remained sequestered inside GCAP1 in both Ca(2+)-bound and Mg(2+)-bound states. This mutant displayed much higher affinity for the cyclase but reduced Ca(2+) sensitivity of the cyclase regulation. The L176F substitution improved affinity of myristoylated and non-acylated GCAP1 for the cyclase but simultaneously reduced the affinity of Ca(2+) binding to EF-hand 4 and Ca(2+) sensitivity of the cyclase regulation by acylated GCAP1. The replacement of amino acids near both ends of the myristoyl moiety (Leu(80) and Val(180)) minimally affected regulatory properties of GCAP1. N-Lauryl- and N-myristoyl-GCAP1 activated RetGC in a similar fashion. Thus, protein interactions with the central region of the fatty acyl chain optimize GCAP1 binding to RetGC and maximize activation of the cyclase. We propose a dynamic connection (or "tug") between the fatty acyl group and EF-hand 4 via the C-terminal helix that attenuates the efficiency of RetGC activation in exchange for optimal Ca(2+) sensitivity.

Molecular structure and target recognition of neuronal calcium sensor proteins.

BACKGROUND: Neuronal calcium sensor (NCS) proteins, a sub-branch of the calmodulin superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulate synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression. SCOPE OF REVIEW: Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase, NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. MAJOR CONCLUSIONS: We propose the idea that N-terminal myristoylation is critical for shaping each NCS family member into a unique structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues, thereby exposing a distinctive ensemble of hydrophobic residues to associate specifically with a particular physiological target. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signaling.

Structure of a Ca2+-myristoyl switch protein that controls activation of a phosphatidylinositol 4-kinase in fission yeast.

Neuronal calcium sensor (NCS) proteins transduce Ca2+ signals and are highly conserved from yeast to humans. We determined NMR structures of the NCS-1 homolog from fission yeast (Ncs1), which activates a phosphatidylinositol 4-kinase. Ncs1 contains an α-NH2-linked myristoyl group on a long N-terminal arm and four EF-hand motifs, three of which bind Ca2+, assembled into a compact structure. In Ca2+-free Ncs1, the N-terminal arm positions the fatty acyl chain inside a cavity near the C terminus. The C14 end of the myristate is surrounded by residues in the protein core, whereas its amide-linked (C1) end is flanked by residues at the protein surface. In Ca2+-bound Ncs1, the myristoyl group is extruded (Ca2+-myristoyl switch), exposing a prominent patch of hydrophobic residues that specifically contact phosphatidylinositol 4-kinase. The location of the buried myristate and structure of Ca2+-free Ncs1 are quite different from those in other NCS proteins. Thus, a unique remodeling of each NCS protein by its myristoyl group, and Ca2+-dependent unmasking of different residues, may explain how each family member recognizes distinct target proteins.

Structural basis for hydration dynamics in radical stabilization of bilin reductase mutants.

Heme-derived linear tetrapyrroles (phytobilins) in phycobiliproteins and phytochromes perform critical light-harvesting and light-sensing roles in oxygenic photosynthetic organisms. A key enzyme in their biogenesis, phycocyanobilin:ferredoxin oxidoreductase (PcyA), catalyzes the overall four-electron reduction of biliverdin IXalpha to phycocyanobilin--the common chromophore precursor for both classes of biliproteins. This interconversion occurs via semireduced bilin radical intermediates that are profoundly stabilized by selected mutations of two critical catalytic residues, Asp105 and His88. To understand the structural basis for this stabilization and to gain insight into the overall catalytic mechanism, we report the high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. PCC 6803 PcyA in the initial oxidized and one-electron reduced radical states. Unlike wild-type PcyA, both mutants possess a bilin-interacting axial water molecule that is ejected from the active site upon formation of the enzyme-bound neutral radical complex. Structural studies of both mutants also show that the side chain of Glu76 is unfavorably located for D-ring vinyl reduction. On the basis of these structures and companion (15)N-(1)H long-range HMQC NMR analyses to assess the protonation state of histidine residues, we propose a new mechanistic scheme for PcyA-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

Effects of Ca2+, Mg2+, and myristoylation on guanylyl cyclase activating protein 1 structure and stability.

Guanylyl cyclase activating protein 1 (GCAP1), a member of the neuronal calcium sensor (NCS) subclass of the calmodulin superfamily, confers Ca(2+)-dependent activation of retinal guanylyl cylcase (RetGC) during phototransduction in vision. Here we analyze the energetics of Ca(2+) and Mg(2+) binding to the individual EF-hands, characterize metal-induced conformational changes, and evaluate structural effects of myristoylation as studied by isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). GCAP1 binds cooperatively to Ca(2+) at EF3 and EF4 (DeltaH(EF3) = -3.5 kcal/mol, and DeltaH(EF4) = -0.9 kcal/mol) with nanomolar affinity (K(EF3) = 80 nM, and K(EF4) = 200 nM), and a third Ca(2+) binds entropically at EF2 (DeltaH(EF2) = 3.1 kcal/mol, and K(EF2) = 0.9 microM). GCAP1 binds functionally to Mg(2+) at EF2 (DeltaH(EF2) = 4.3 kcal/mol, and K(EF2) = 0.7 mM) required for RetGC activation. Ca(2+) and/or Mg(2+) binding to GCAP1 dramatically alters DSC and NMR spectra, indicating metal-induced protein conformational changes in EF2, EF3, and EF4. Myristoylation of GCAP1 does not significantly alter its metal binding energetics or NMR spectra, suggesting that myristoylation does not influence the structure of the metal-binding EF-hands. Myristoylation also has almost no effect on protein folding stability measured by DSC. NMR resonances of myristate attached to GCAP1 are exchange-broadened, upfield-shifted, and insensitive to Ca(2+), consistent with the myristoyl group being sequestered inside the protein as seen in the crystal structure. We conclude that the protein environment near the myristate is not influenced by Mg(2+) or Ca(2+) binding but instead is constitutively dynamic and may play a role in promoting interactions of GCAP1 with the cyclase.

Selective protein patterning based on the micro-structured organosilane self-assembled monolayer by vacuum ultraviolet light lithography.

We have succeeded to immobilize fluorescent proteins selectively using a micro-structured organosilane self-assembled monolayer as a template. An organosilane layer with amino terminal group was formed on a thermally oxidized Si wafer by liquid-phase method and then was pattern-etched by vacuum ultraviolet light (VUV). The second organosilane layer with thiol terminal group was deposited on the etched area by chemical vapor surface modification method (CVSM). These micro-structured organosilane layer containing two reactive terminal groups were chemically modified using bi-functional linkers. Two kinds of fluorescent protein, Enhanced Cyan Fluorescent Protein (ECFP) and R-phycoerythrin were selectively immobilized on the chemically modified surface.

Retinal guanylyl cyclase activating protein 1 forms a functional dimer.

Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg2+-bound activator and Ca2+-bound inhibitor states (RMSD < 2.0 Å). The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations (H19R, Y22D, F73E, and V77E) at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.

Chemical shift assignments of retinal degeneration 3 protein (RD3).

Retinal degeneration 3 protein (RD3) binds to retinal membrane guanylyl cyclase (RetGC) and suppresses the basal activity of RetGC in photoreceptor cells that opposes the allosteric activation of the cyclase by GCAP proteins. Mutations in RD3 that disrupt its inhibition of RetGC are implicated in human retinal degenerative disorders. Here we report both backbone and sidechain NMR assignments for the RD3 protein (BMRB accession no. 27305).

Engineered lanthanide-binding metallohomeodomains: designing folded chimeras by modular turn substitution.

A series of chimeric metallohomeodomains are described, engineered by rational design of a flexible Ca/Ln binding site into a DNA-binding scaffold. A modular turn-substitution approach was used to create proteins that both bind DNA and lanthanide ions, while retaining the secondary structure of the full homeodomain (determined by circular dichroism [CD]). Four similar metallohomeodomains were designed (C1-C4), their structural stability predicted by molecular dynamics (MD) simulation of loop-mutations into the known homeodomain structure, and each designed protein cloned, expressed, and purified using standard molecular biology techniques. Two of the four loop insertions resulted in folded, metal- and DNA-binding proteins (EuC2 Kd = 2.1 +/- 0.4 microM; EuC4 Kd = 3.2 +/- 1.0 microM). These results show the successful incorporation of a metal site into a full protein domain, without compromising long-range structure. This is an important achievement in biomolecular design, as it provides a critical starting point for exploring metallonuclease function and substrate accessibility in a well-organized chimeric protein domain (rather than only in small HTH peptide systems).

Structure and Calcium Binding Properties of a Neuronal Calcium-Myristoyl Switch Protein, Visinin-Like Protein 3.

Visinin-like protein 3 (VILIP-3) belongs to a family of Ca2+-myristoyl switch proteins that regulate signal transduction in the brain and retina. Here we analyze Ca2+ binding, characterize Ca2+-induced conformational changes, and determine the NMR structure of myristoylated VILIP-3. Three Ca2+ bind cooperatively to VILIP-3 at EF2, EF3 and EF4 (KD = 0.52 µM and Hill slope of 1.8). NMR assignments, mutagenesis and structural analysis indicate that the covalently attached myristoyl group is solvent exposed in Ca2+-bound VILIP-3, whereas Ca2+-free VILIP-3 contains a sequestered myristoyl group that interacts with protein residues (E26, Y64, V68), which are distinct from myristate contacts seen in other Ca2+-myristoyl switch proteins. The myristoyl group in VILIP-3 forms an unusual L-shaped structure that places the C14 methyl group inside a shallow protein groove, in contrast to the much deeper myristoyl binding pockets observed for recoverin, NCS-1 and GCAP1. Thus, the myristoylated VILIP-3 protein structure determined in this study is quite different from those of other known myristoyl switch proteins (recoverin, NCS-1, and GCAP1). We propose that myristoylation serves to fine tune the three-dimensional structures of neuronal calcium sensor proteins as a means of generating functional diversity.

1H, 15N, and 13C chemical shift assignments of cyanobacteriochrome NpR6012g4 in the red-absorbing dark state.

Cyanobacteriochrome (CBCR) photosensory proteins are phytochrome homologs using bilin chromophores for light sensing across the visible spectrum. NpR6012g4 is a CBCR from Nostoc punctiforme that serves as a model for a widespread CBCR subfamily with red/green photocycles. We report NMR chemical shift assignments for both the protein backbone and side-chain resonances of the red-absorbing dark state of NpR6012g4 (BMRB no. 26582).

1H, 13C, and 15N chemical shift assignments of cyanobacteriochrome NpR6012g4 in the green-absorbing photoproduct state.

Cyanobacteriochromes (CBCRs) are cyanobacterial photosensory proteins with a tetrapyrrole (bilin) chromophore that belong to the phytochrome superfamily. Like phytochromes, CBCRs photoconvert between two photostates with distinct spectral properties. NpR6012g4 from Nostoc punctiforme is a model system for widespread CBCRs with conserved red/green photocycles. Atomic-level structural information for the photoproduct state in this subfamily is not known. Here, we report NMR backbone chemical shift assignments of the light-activated state of NpR6012g4 (BMRB no. 26577) as a first step toward determining its atomic resolution structure.