DNA modifications: Biomarkers for the exposome?

The concept of the exposome is the encompassing of all the environmental exposures, both exogenous and endogenous, across the life course. Many, if not all, of these exposures can result in the generation of reactive species, and/or the modulation of cellular processes, that can lead to a breadth of modifications of DNA, the nature of which may be used to infer their origin. Because of their role in cell function, such modifications have been associated with various major human diseases, including cancer, and so their assessment is crucial. Historically, most methods have been able to only measure one or a few DNA modifications at a time, limiting the information available. With the development of DNA adductomics, which aims to determine the totality of DNA modifications, a far more comprehensive picture of the DNA adduct burden can be gained. Importantly, DNA adductomics can facilitate a "top-down" investigative approach whereby patterns of adducts may be used to trace and identify the originating exposure source. This, together with other 'omic approaches, represents a major tool for unraveling the complexities of the exposome and hence allow a better a understanding of the environmental origins of disease.

Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality.

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only ∼2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl ß-d-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

Conodipine-P1-3, the First Phospholipases A2 Characterized from Injected Cone Snail Venom.

The phospholipase A2 (PLA2s) superfamily are ubiquitous small enzymes that catalyze the hydrolysis of phospholipids at the sn-2 ester bond. PLA2s in the venom of cone snails (conodipines, Cdpi) are composed of two chains termed as alpha and beta subunits. Conodipines are categorized within the group IX of PLA2s. Here we describe the purification and biochemical characterization of three conodipines (Cdpi-P1, -P2 and -P3) isolated from the injected venom of Conus purpurascens Using proteomics methods, we determined the full sequences of all three conodipines. Conodipine-P1-3 have conserved consensus catalytic domain residues, including the Asp/His dyad. Additionally, these enzymes are expressed as a mixture of proline hydroxylated isoforms. The activities of the native Conodipine-Ps were evaluated by conventional colorimetric and by MS-based methods, which provide the first detailed cone snail venom conodipine activity monitored by mass spectrometry. Conodipines can have medicinal applications such inhibition of cancer proliferation, bacterial and viral infections among others.

LC-MS/MS based detection and characterization of covalent glutathione modifications formed by reactive drug of abuse metabolites.

Conjugation with the tripeptide glutathione (GSH) is a common mechanism of detoxification of many endogenous and exogenous compounds. This phenomenon typically occurs through the formation of a covalent bond between the nucleophilic free thiol moiety of GSH and an electrophilic site on the compound of interest. While GSH adducts have been identified for many licit drugs, there is a lack of information on the ability of drugs of abuse to adduct GSH. The present study utilized a metabolic assay with GSH as a nucleophilic trapping agent to bind reactive drug metabolites formed in situ. Extracted ion MS spectra were collected via LC-QqQ-MS/MS for all potentially significant ions and examined for fragmentation common to GSH-containing compounds, followed by confirmation of adduction and structural characterization performed by LC-QTOF-MS/MS. In addition to the two positive controls, of the 14 drugs of abuse tested, 10 exhibited GSH adduction, with several forming multiple adducts, resulting in a total of 22 individual identified adducts. A number of these are previously unreported in the literature, including those for diazepam, naltrexone, oxycodone and Δ9-THC.

Definition of the R-superfamily of conotoxins: Structural convergence of helix-loop-helix peptidic scaffolds.

The F14 conotoxins define a four-cysteine, three-loop conotoxin scaffold that produce tightly folded structures held together by two disulfide bonds with a CCCC arrangement (conotoxin framework 14). Here we describe the precursors of the F14 conotoxins from the venom of Conus anabathrum and Conus villepinii. Using transcriptomic and cDNA cloning analysis, the full-length of the precursors of flf14a and flf14b from the transcriptome of C. anabathrum revealed a unique signal sequence that defines the new conotoxin R-superfamily. Using the signal sequence as a primer, we cloned seven additional previously undescribed toxins of the R-superfamily from C. villepinii. The propeptide regions of the R-conotoxins are unusually long and with prevalent proline residues in repeating pentads which qualifies them as Pro-rich motifs (PRMs), which can be critical for protein-protein interactions or they can be cleaved to release short linear peptides that may be part of the envenomation mélange. Additionally, we determined the three-dimensional structure of vil14a by solution 1H-NMR and found that the structure of this conotoxin displays a cysteine-stabilized α-helix-loop-helix (Cs α/α) fold. The structure is well-defined over the helical regions (backbone RMSD for residues 2-13 and 17-26 is 0.63 ± 0.14 Å), with conformational flexibility in the triple Gly region of the second loop as well as the N- and C- termini. Structurally, the F14 conotoxins overlap with the Cs α/α scorpion toxins and other peptidic natural products, and in spite of their different exogenomic origins, there is convergence into this scaffold from several classes of living organisms that express these peptides.

Structural plasticity of mini-M conotoxins - expression of all mini-M subtypes by Conus regius.

The mini-M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC-C-C-CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini-M conotoxins from the venom of Conus regius, a Western Atlantic worm-hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini-Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D-NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi-turn scaffold. The structural diversity found within mini-M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications. DATABASES: Sequences are available in GenBank under accession numbers MF588935-MF588952. Structural data are available in the RCSB protein database under the accession code 6BX9.

Proteomic Analysis of Thiol Modifications and Assessment of Structural Changes in Hemoglobin Induced by the Aniline Metabolites N-Phenylhydroxylamine and Nitrosobenzene.

MS-based proteomic analysis was combined with in silico quantum mechanical calculations to improve understanding of protein adduction by N-phenylhydroxylamine (PhNHOH) and nitrosobenzene (NOB), metabolic products of aniline. In vitro adduction of model peptides containing nucleophilic sidechains (Cys, His, and Lys) and selected proteins (bovine and human hemoglobin and ß-lactoglobulin-A) were characterized. Peptide studies identified the Cys thiolate as the most reactive nucleophile for these metabolites, a result consistent with in silico calculations of reactivity parameters. For PhNHOH, sulfinamides were identified as the primary adduction products, which were stable following tryptic digestion. Conversely, reactions with NOB yielded an additional oxidized adduct, the sulfonamide. In vitro exposure of human whole blood to PhNHOH and NOB demonstrated that only sulfinamides were formed. In addition to previously reported adduction of ß93Cys of human Hb, two novel sites of adduction were found; α104Cys and ß112Cys. We also report CD and UV-Vis spectroscopy studies of adducted human Hb that revealed loss of α-helical content and deoxygenation. The results provide additional understanding of the covalent interaction of aromatic amine metabolites with protein nucleophiles.

Isolation and characterization of Conohyal-P1, a hyaluronidase from the injected venom of Conus purpurascens.

Hyaluronidases are ubiquitous enzymes commonly found in venom and their main function is to degrade hyaluran, which is the major glycosaminoglycan of the extracellular matrix in animal tissues. Here we describe the purification and characterization of a 60kDa hyaluronidase found in the injected venom from Conus purpurascens, Conohyal-P1. Using a combined strategy based on transcriptomic and proteomic analysis, we determined the Conohyal-P1 sequence. Conohyal-P1 has conserved consensus catalytic and positioning domain residues characteristic of hyaluronidases and a C-terminus EGF-like domain. Additionally, the enzyme is expressed as a mixture of glycosylated isoforms at five asparagine sites. The activity of the native Conohyal-P1 was assess MS-based methods and confirmed by classical turbidimetric methods. The MS-based assay is particularly sensitive and provides the first detailed analysis of a venom hyaluronidase activity monitored with this method. The discovery of new hyaluronidases and the development of techniques to evaluate their performance can advance several therapeutic procedures, as these enzymes are widely used for enhanced drug delivery applications. BIOLOGICAL SIGNIFICANCE: Cone snail venom is a remarkable source of therapeutically important molecules, as is the case of conotoxins, which have undergone extensive clinical trials for several applications. In addition to the conotoxins, a large array of proteins have been reported in the venom of several species of cone snails, including enzymes that were found in dissected and injected Conus venom. Here we describe the isolation and characterization of the hyaluronidase Conohyal-P1 from the injected venom of C. purpurascens. We employed a combined transcriptomic and proteomic analysis to obtain the full sequence of this hyaluronidase. The activity of Conohyal-P1 was assessed by a mass spectrometry-based method, which provide the first detailed venom hyaluronidase activity analysis monitored by mass spectrometry allowing the visualization of the substrate degradation by the enzyme.

Correlation of transducin photoaffinity labeling with the specific formation of intermolecular disulfide linkages in its α-subunit.

Transducin (T) is a heterotrimer of Tα, Tß, and Tγ subunits. In the presence of light-activated rhodopsin, 8-azidoguanosine triphosphate (8-N3GTP) was covalently incorporated into T in a UV-light photodependent manner, with a low stoichiometry of 0.02 mol of 8-N3GTP per mol of T. Although Tα was preferentially labeled by 8-N3GTP, Tß and Tγ were also modified. Photolabeling of T was specifically inhibited by GDP and GTP, but not by ß,γ-imido-guanosine 5'-triphosphate (GMP-PNP), indicating that 8-N3GTP was modifying the GDP binding site of the holoenzyme. This was consistent with the observation that the photoaffinity probe was completely hydrolyzed to 8-N3GDP by T activated by illuminated rhodopsin. The formation of intermolecular disulfide associations in T was also determined because photolabeling of T was performed under non-reducing conditions. We established that Cys-347 of Tα was the major residue involved in the formation of disulfide-linked T oligomers. Other cysteines of Tα, such as Cys-321, also participated in the formation of disulfide bonds, revealing a complex pattern of intermolecular disulfide cross-links that led to the polymerization of T. The spontaneous generation of these cystines in Tα inhibited the light-dependent GTPase and GMP-PNP binding activities of T. A model was constructed illustrating that when two heterotrimers dimerize through the formation of disulfide bridges between the Cys-347 of their Tα subunits, the guanine ring of the 8-N3GDP bound to one T molecule might approach to the Tßγ-complex of the other heterotrimer. This model provides an explanation for the additional photolabeling of Tß and Tγ by 8-N3GTP.

High molecular weight components of the injected venom of fish-hunting cone snails target the vascular system.

The venom of marine cone snails is a rich source of pharmacotherapeutic compounds with striking target specificity and functional diversity. Small, disulfide-rich peptide toxins are the most well characterized active compounds in cone snail venom. However, reports on the presence of larger polypeptides have recently emerged. The majority of these studies have focused on the content of the dissected venom gland rather than the injected venom itself. Recent breakthroughs in the sensitivity of protein and nucleotide sequencing techniques allow for the exploration of the proteomic diversity of injected venom. Using mass spectrometric analysis of injected venoms of the two fish-hunting cone snails Conus purpurascens and Conus ermineus, we demonstrate the presence of angiotensin-converting enzyme-1 (ACE-1) and endothelin converting enzyme-1 (ECE-1), metalloproteases that activate potent vasoconstrictive peptides. ACE activity was confirmed in the venom of C. purpurascens and was significantly reduced in venom preincubated with the ACE inhibitor captopril. Reverse-transcription PCR demonstrated that these enzymes are expressed in the venom glands of other cone snail species with different prey preferences. These findings strongly suggest that cone snails employ compounds that cause disruption of cardiovascular function as part of their complex envenomation strategy, leading to the enhancement of neurotropic peptide toxin activity. BIOLOGICAL SIGNIFICANCE: To our knowledge, this is the first study to show the presence of ACE and ECE in the venom of cone snails. Identification of these vasoactive peptide-releasing proteases in the injected venoms of two fish-hunting cone snails highlights their role in envenomation and enhances our understanding of the complex hunting strategies utilized by these marine predators. Our findings on the expression of these enzymes in other cone snail species suggests an important biological role of ACE and ECE in these animals and points towards recruitment into venom from general physiological processes.

Comparative analysis of proteases in the injected and dissected venom of cone snail species.

The venom of cone snails has been the subject of intense studies because it contains small neuroactive peptides of therapeutic value. However, much less is known about their larger proteins counterparts and their role in prey envenomation. Here, we analyzed the proteolytic enzymes in the injected venom of Conus purpurascens and Conus ermineus (piscivorous), and the dissected venom of C. purpurascens, Conus marmoreus (molluscivorous) and Conus virgo (vermivorous). Zymograms show that all venom samples displayed proteolytic activity on gelatin. However, the electrophoresis patterns and sizes of the proteases varied considerably among these four species. The protease distribution also varied dramatically between the injected and dissected venom of C. purpurascens. Protease inhibitors demonstrated that serine and metalloproteases are responsible for the gelatinolytic activity. We found fibrinogenolytic activity in the injected venom of C. ermineus suggesting that this venom might have effects on the hemostatic system of the prey. Remarkable differences in protein and protease expression were found in different sections of the venom duct, indicating that these components are related to the storage granules and that they participate in venom biosynthesis. Consequently, different conoproteases play major roles in venom processing and prey envenomation.

Unraveling the peptidome of the South African cone snails Conus pictus and Conus natalis.

Venoms from cone snails (genus Conus) can be seen as an untapped cocktail of biologically active compounds, being increasingly recognized as an emerging source of peptide-based therapeutics. Cone snails are considered to be specialized predators that have evolved the most sophisticated peptide chemistry and neuropharmacology system for their own biological purposes by producing venoms which contains a structural and functional diversity of neurotoxins. These neurotoxins or conotoxins are often small cysteine-rich peptides which have shown to be highly selective ligands for a wide range of ion channels and receptors. Local habitat conditions have constituted barriers preventing the spreading of Conus species occurring along the coast of South Africa. Due to their scarceness, these species remain, therefore, extremely poorly studied. In this work, the venoms of two South African cone snails, Conus pictus, a vermivorous snail and Conus natalis, a molluscivorous snail, have been characterized in depth. In total, 26 novel peptides were identified. Comparing the venoms of both snails, interesting differences were observed regarding venom composition and molecular characteristics of these components.

A natural point mutation changes both target selectivity and mechanism of action of sea anemone toxins.

APETx3, a novel peptide isolated from the sea anemone Anthopleura elegantissima, is a naturally occurring mutant from APETx1, only differing by a Thr to Pro substitution at position 3. APETx1 is believed to be a selective modulator of human ether-á-go-go related gene (hERG) potassium channels with a K(d) of 34 nM. In this study, APETx1, 2, and 3 have been subjected to an electrophysiological screening on a wide range of 24 ion channels expressed in Xenopus laevis oocytes: 10 cloned voltage-gated sodium channels (Na(V) 1.2-Na(V)1.8, the insect channels DmNa(V)1, BgNa(V)1-1a, and the arachnid channel VdNa(V)1) and 14 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, K(V)7.2, K(V)7.4, hERG, and the insect channel Shaker IR). Surprisingly, the Thr3Pro substitution results in a complete abolishment of APETx3 modulation on hERG channels and provides this toxin the ability to become a potent (EC(50) 276 nM) modulator of voltage-gated sodium channels (Na(V)s) because it slows down the inactivation of mammalian and insect Na(V) channels. Our study also shows that the homologous toxins APETx1 and APETx2 display promiscuous properties since they are also capable of recognizing Na(V) channels with IC(50) values of 31 nM and 114 nM, respectively, causing an inhibition of the sodium conductance without affecting the inactivation. Our results provide new insights in key residues that allow these sea anemone toxins to recognize distinct ion channels with similar potency but with different modulatory effects. Furthermore, we describe for the first time the target promiscuity of a family of sea anemone toxins thus far believed to be highly selective.

Pc16a, the first characterized peptide from Conus pictus venom, shows a novel disulfide connectivity.

A novel conotoxin, pc16a, was isolated from the venom of Conus pictus. This is the first peptide characterized from this South-African cone snail and it has only 11 amino acid residues, SCSCKRNFLCC*, with the rare cysteine framework XVI and a monoisotopic mass of 1257.6Da. Two peptides were synthesized with two possible conformations: globular (pc16a_1) and ribbon (pc16a_2). pc16a_1 co-eluted with the native peptide, which indicates a disulfide connectivity I-III, II-IV. The structure of pc16a_1 was determined by NMR. Both synthetic peptides were used to elucidate the biological activity. Bioassays were performed on crickets, ghost shrimps, larvae of the mealworm beetle and mice, but no effect was seen. Using two-electrode voltage clamp, a range of voltage-gated ion channels (Na(v) and K(v)) and nicotinic acetylcholine receptors were screened, but again no activity was found. Hence, the specific target of pc16a still remains to be discovered.

9.3 KDa components of the injected venom of Conus purpurascens define a new five-disulfide conotoxin framework.

The 83-residue conopeptide (p21a) and its corresponding nonhydroxylated analog were isolated from the injected venom of Conus purpurascens. The complete conopeptide sequences were derived from Edman degradation sequencing of fragments from tryptic, chymotryptic and cyanogen bromide digestions, p21a has a unique, 10-cystine/5-disulfide 7-loop framework with extended 10-residue N-terminus and a 5-residue C-terminus tails, respectively. p21a has a 48% sequence homology with a recently described 13-cystine conopeptide, con-ikot-ikot, isolated from the dissected venom of the fish-hunting snail Conus striatus. However, unlike con-ikot-ikot, p21a does not form a dimer of dimers. MALDI-TOF mass spectrometry suggests that p21a may form a noncovalent dimer. p21a is an unusually large conotoxin and in so far is the largest isolated from injected venom. p21a provides evidence that the Conus venom arsenal includes larger molecules that are directly injected into the prey. Therefore, cone snails can utilize toxins that are comparable in size to the ones commonly found in other venomous animals.

Functional hypervariability and gene diversity of cardioactive neuropeptides.

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

Aproximaciones de bioquímica clásica al estudio de la relación entre la estructura y la función de la rodopsina Y / Approaches to the study of classical biochemistry of the relationship between structure and function of the rhodopsin

La proteína fotorreceptora rodopsina (R) fue extraída de los segmentos externos de los bastoncillos de retinas bovinas con el detergente n-dodecil β-D-maltósido (DM) y purificada a homogeneidad mediante cromatografía de afinidad. El entrecruzamiento químico de la R y de la rodopsina fotoactivada (R*) con los agentes bifuncionales sulfo-succinimidilo 4-(N-maleimidometilo) ciclohexano-1-carboxilato (sulfo-SMCC) o m-maleimidobenzoilo-N-hidroxisuccinimido ester, sugirieron la naturaleza oligomérica de la proteína fotorreceptora. La caracterización de los parámetros hidrodinámicos de la R y la R* en presencia de 0.1% DM, mediante cromatografía de exclusión molecular y sedimentación sobre gradientes de sacarosa, permitió estimar los tamaños de los complejos R:DM y R*: DM. Los resultados concuerdan con una estructura cuaternaria dimérica tanto para la R como para la R*. La R entrecruzada con sulfo-SMCC, en presencia de luz, fue estabilizada en un fotointermediario que absorbió a ~ 470 nm. Experimentos de proteólisis con termolísina sobre los dímeros nativos de R y sobre los monómeros de R generados por medio del uso de altas concentraciones de DM, complementados con estudios de modelaje basados en la estructura cristalina reportada de la proteína, sugirieron que el reactivo sulfo-SMCC generó un entrecruzamiento intramolecular entre la Cys140 y la Lys248 de la R, el cual posiblemente es el responsable de la incapacidad de la proteína de sufrir el cambio conformacional requerido para llegar a su estado fotoactivado.

A vasopressin/oxytocin-related conopeptide with gamma-carboxyglutamate at position 8.

Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated gamma-conopressin-vil, has the sequence CLIQDCPgammaG* (gamma is gamma-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is gamma-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native gamma-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the gamma-carboxyglutamate residue. However, the negatively charged residues in the sequence of gamma-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.

A novel conotoxin framework with a helix-loop-helix (Cs alpha/alpha) fold.

Venomous predatory animals, such as snakes, spiders, scorpions, sea anemones, and cone snails, produce a variety of highly stable cystine-constrained peptide scaffolds as part of their neurochemical strategy for capturing prey. Here we report a new family of four-cystine, three-loop conotoxins (designated framework 14). Three peptides of this family (flf14a-c) were isolated from the venom of Conus floridanus floridensis, and one (vil14a) was isolated from the venom of Conus villepinii, two worm-hunting Western Atlantic cone snail species. The primary structure for these peptides was determined using Edman degradation sequencing, and their cystine pairing was assessed by limited hydrolysis with a combination of CNBr and chymotrypsin under nonreducing, nonalkylating conditions in combination with MALDI-TOF MS analysis of the resulting peptidic fragments. CD spectra and nanoNMR spectroscopy of these conotoxins directly isolated from the cone snails revealed a highly helical secondary structure for the four conotoxins. Sequence-specific nanoNMR analysis at room temperature revealed a well-defined helix-loop-helix tertiary structure that resembles that of the Cs alpha/alpha scorpion toxins kappa-hefutoxin, kappa-KTx1.3, and Om-toxins, which adopt a stable three-dimensional fold where the two alpha-helices are linked by the two disulfide bridges. One of these conotoxins (vil14a) has a Lys/Tyr dyad, separated by approximately 6A, which is a conserved structural feature in K(+) channel blockers. The presence of this framework in scorpions and in cone snails indicates a common molecular imprint in the venom of apparently unrelated predatory animals and suggests a common ancestral genetic origin.

Chemical modification of transducin with dansyl chloride hinders its binding to light-activated rhodopsin.

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.