Occurrence and evolutionary conservation analysis of α-helical cationic amphiphilic segments in the human proteome.

The existence of encrypted fragments with antimicrobial activity in human proteins has been thoroughly demonstrated in the literature. Recently, algorithms for the large-scale identification of these segments in whole proteomes were developed, and the pervasiveness of this phenomenon was stated. These algorithms typically mine encrypted cationic and amphiphilic segments of proteins, which, when synthesized as individual polypeptide sequences, exert antimicrobial activity by membrane disruption. In the present report, the human reference proteome was submitted to the software kamal for the uncovering of protein segments that correspond to putative intragenic antimicrobial peptides (IAPs). The assessment of the identity of these segments, frequency, functional classes of parent proteins, structural relevance, and evolutionary conservation of amino acid residues within their corresponding proteins was conducted in silico. Additionally, the antimicrobial and anticancer activity of six selected synthetic peptides was evaluated. Our results indicate that cationic and amphiphilic segments can be found in 2% of all human proteins, but are more common in transmembrane and peripheral membrane proteins. These segments are surface-exposed basic patches whose amino acid residues present similar conservation scores to other residues with similar solvent accessibility. Moreover, the antimicrobial and anticancer activity of the synthetic putative IAP sequences was irrespective to whether these are associated to membranes in the cellular setting. Our study discusses these findings in light of the current understanding of encrypted peptide sequences, offering some insights into the relevance of these segments to the organism in the context of their harboring proteins or as separate polypeptide sequences.

Release of immunomodulatory peptides at bacterial membrane interfaces as a novel strategy to fight microorganisms.

Cationic and amphiphilic peptides can be used as homing devices to accumulate conjugated antibiotics to bacteria-enriched sites and promote efficient microbial killing. However, just as important as tackling bacterial infections, is the modulation of the immune response in this complex microenvironment. In the present report, we designed a peptide chimaera called Chim2, formed by a membrane-active module, an enzyme hydrolysis site and a formyl peptide receptor 2 (FPR2) agonist. This molecule was designed to adsorb onto bacterial membranes, promote their lysis, and upon hydrolysis by local enzymes, release the FPR2 agonist sequence for activation and recruitment of immune cells. We synthesized the isolated peptide modules of Chim2 and characterized their biological activities independently and as a single polypeptide chain. We conducted antimicrobial assays, along with other tests aiming at the analyses of the cellular and immunological responses. In addition, assays using vesicles as models of eukaryotic and prokaryotic membranes were conducted and solution structures of Chim2 were generated by 1H NMR. Chim2 is antimicrobial, adsorbs preferentially to negatively charged vesicles while adopting an α-helix structure and exposes its disorganized tail to the solvent, which facilitates hydrolysis by tryptase-like enzymes, allowing the release of the FPR2 agonist fragment. This fragment was shown to induce accumulation of the cellular activation marker, lipid bodies, in mouse macrophages and the release of immunomodulatory interleukins. In conclusion, these data demonstrate that peptides with antimicrobial and immunomodulatory activities can be considered for further development as drugs.

Probing human proteins for short encrypted antimicrobial peptides reveals Hs10, a peptide with selective activity for gram-negative bacteria.

BACKGROUND: Some cationic and amphiphilic α-helical segments of proteins adsorb to prokaryotic membranes when synthesized as individual polypeptide sequences, resulting in broad and potent antimicrobial activity. However, amphiphilicity, a determinant physicochemical property for peptide-membrane interactions, can also be observed in some ß-sheets. METHODS: The software Kamal was used to scan the human reference proteome for short (7-11 amino acid residues) cationic and amphiphilic protein segments with the characteristic periodicity of ß-sheets. Some of the uncovered peptides were chemically synthesized, and antimicrobial assays were conducted. Biophysical techniques were used to probe the molecular interaction of one peptide with phospholipid vesicles, lipopolysaccharides (LPS) and the bacterium Escherichia coli. RESULTS: Thousands of compatible segments were found in human proteins, five were synthesized, and three presented antimicrobial activity in the micromolar range. Hs10, a nonapeptide fragment of the Complement C3 protein, could inhibit only the growth of tested Gram-negative microorganisms, presenting also little cytotoxicity to human fibroblasts. Hs10 interacted with LPS while transitioning from an unstructured segment to a ß-sheet and increased the hydrodynamic radius of LPS particles. This peptide also promoted morphological alterations in E. coli cells. CONCLUSIONS: Data presented herein introduce yet another molecular template to probe proteins in search for encrypted membrane-active segments and demonstrates that, using this approach, short peptides with low cytotoxicity and high selectivity to prokaryotic cells might be obtained. GENERAL SIGNIFICANCE: This work widens the biotechnological potential of the human proteome as a source of antimicrobial peptides with application in human health.

Novel disintegrin-like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus.

Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

Identification of Differential N-Glycan Compositions in the Serum and Tissue of Colon Cancer Patients by Mass Spectrometry.

Colorectal cancer (CRC) ranks second as the leading cause of cancer-related deaths worldwide. N-glycosylation is one of the most common posttranslational protein modifications. Therefore, we studied the total serum N-glycome (TSNG) of 13 colon cancer patients compared to healthy controls using MALDI-TOF/MS and LC-MS. N-glycosylation of cancer tumor samples from the same cohort were further quantified using a similar methodology. In total, 23 N-glycan compositions were down-regulated in the serum of colon cancer patients, mostly galactosylated forms whilst the mannose-rich HexNAc2Hex7, the fucosylated bi-antennary glycan HexNAc4Hex5Fuc1NeuAc2, and the tetra-antennary HexNAc6Hex7NeuAc3 were up-regulated in serum. Hierarchical clustering analysis of TSNG correctly singled out 85% of the patients from controls. Albeit heterogenous, N-glycosylation of tumor samples showed overrepresented oligomannosidic, bi-antennary hypogalactosylated, and branched compositions related to normal colonic tissue, in both MALDI-TOF/MS and LC-MS analysis. Moreover, compositions found upregulated in tumor tissue were mostly uncorrelated to compositions in serum of cancer patients. Mass spectrometry-based N-glycan profiling in serum shows potential in the discrimination of patients from healthy controls. However, the compositions profile in serum showed no parallel with N-glycans in tumor microenvironment, which suggests a different origin of compositions found in serum of cancer patients.

Phylloseptin-1 is Leishmanicidal for Amastigotes of Leishmaniaamazonensis Inside Infected Macrophages.

Leishmania protozoans are the causal agents of neglected diseases that represent an important public health issue worldwide. The growing occurrence of drug-resistant strains of Leishmania and severe side effects of available treatments represent an important challenge for the leishmaniases treatment. We have previously reported the leishmanicidal activity of phylloseptin-1 (PSN-1), a peptide found in the skin secretion of Phyllomedusaazurea (=Pithecopus azureus), against Leishmaniaamazonensis promastigotes. However, its impact on the amastigote form of L. amazonensis and its impact on infected macrophages are unknown. In this work, we evaluated the effects of PSN-1 on amastigotes of L. amazonensis inside macrophages infected in vitro. We assessed the production of hydrogen peroxide and nitric oxide, as well as the levels of inflammatory and immunomodulatory markers (TGF-ß, TNF-α and IL-12), in infected and non-infected macrophages treated with PSN-1. Treatment with PSN-1 decreased the number of infected cells and the number of ingested amastigotes per cell when compared with the untreated cells. At 32 µM (64 µg/mL), PSN-1 reduced hydrogen peroxide levels in both infected and uninfected macrophages, whereas it had little effect on NO production or TGF-ß release. The effect of PSN-1 on IL-12 and TNF-α secretion depended on its concentration, but, in general, their levels tended to increase as PSN-1 concentration increased. Further in vitro and in vivo studies are needed to clarify the mechanisms of action of PSN-1 and its interaction with the immune system aiming to develop pharmacological applications.

Intragenic antimicrobial peptides (IAPs) from human proteins with potent antimicrobial and anti-inflammatory activity.

Following the treads of our previous works on the unveiling of bioactive peptides encrypted in plant proteins from diverse species, the present manuscript reports the occurrence of four proof-of-concept intragenic antimicrobial peptides in human proteins, named Hs IAPs. These IAPs were prospected using the software Kamal, synthesized by solid phase chemistry, and had their interactions with model phospholipid vesicles investigated by differential scanning calorimetry and circular dichroism. Their antimicrobial activity against bacteria, yeasts and filamentous fungi was determined, along with their cytotoxicity towards erythrocytes. Our data demonstrates that Hs IAPs are capable to bind model membranes while attaining α-helical structure, and to inhibit the growth of microorganisms at concentrations as low as 1µM. Hs02, a novel sixteen residue long internal peptide (KWAVRIIRKFIKGFIS-NH2) derived from the unconventional myosin 1h protein, was further investigated in its capacity to inhibit lipopolysaccharide-induced release of TNF-α in murine macrophages. Hs02 presented potent anti-inflammatory activity, inhibiting the release of TNF-α in LPS-primed cells at the lowest assayed concentration, 0.1 µM. A three-dimensional solution structure of Hs02 bound to DPC micelles was determined by Nuclear Magnetic Resonance. Our work exemplifies how the human genome can be mined for molecules with biotechnological potential in human health and demonstrates that IAPs are actual alternatives to antimicrobial peptides as pharmaceutical agents or in their many other putative applications.

Identification and characterization of phospholipases A2 from the skin secretion of Pithecopus azureus anuran.

The present work reports the isolation, characterization and the complete sequence of a phospholipase A2 (PLA2) present in the skin secretion of Pithecopus azureus. Among several peptides and small proteins previously described by our group from some species belonging to this amphibian genus (formerly named Phyllomedusa), a 15 kDa N-glycosylated protein showing PLA2 activity was purified, assayed, sequenced and named Pa-PLA2. The Pithecopus azureus skin phospholipase A2 polypeptide chain is composed by 125 amino acid residues linked by seven disulfide bonds and two N-glycosylated sites (N67 and N108). The Pa-PLA2 enzymatic activity was qualitatively evaluated and compared to classical viperid PLA2 showing that both, native and deglycosylated Pa-PLA2 forms, are catalytically functional. The tridimensional molecular model of Pa-PLA2 indicates that the observed glycan moieties are suggestively placed far from the active site of that enzyme and therefore having little or no significant role on the direct interaction of the Pa-PLA2 catalytic pocket and its substrates.

A previously undescribed hexapeptide His-Arg-Phe-Leu-Arg-His-NH2 from amphibian skin secretion shows CO2 and metal biding affinities.

A previously undescribed six residues long peptide His-Arg-Phe-Leu-Arg-His was identified and purified from the skin secretion of the amphibian Phyllomedusa centralis. A synthetic analogue carboxyamidated HRFLRH-NH2 showed structural changes induced by CO2 and metal ions in aqueous solution when analyzed by NMR. The present work reports NMR structures for the carboxyamidated hexapeptide in the presence CO2, Zn2+ and Cd2+, suggesting possible affinity regions on the polypeptide chain for each ligand. The NMR structures were optimized by DFT to identify probable biding sites of these species in the polypeptide structure. To our best knowledge, this is the first time that a putative CO2 binding site is described on a peptide structure obtained in aqueous conditions, at room temperature.

Imaging Mass Spectrometry of Endogenous Polypeptides and Secondary Metabolites from Galls Induced by Root-Knot Nematodes in Tomato Roots.

Nematodes are devastating pests that infect most cultivated plant species and cause considerable agricultural losses worldwide. The understanding of metabolic adjustments induced during plant-nematode interaction is crucial to generate resistant plants or to select more efficient molecules to fight against this pest. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used herein for in situ detection and mapping endogenous polypeptides and secondary metabolites from nematode-induced gall tissue. One of the major critical features of this technique is sample preparation; mainly, the generation of intact sections of plant cells with their rigid cell walls and vacuolated cytoplasm. Our experimental settings allowed us to obtain sections without contamination of exogenous ions or diffusion of molecules and to map the differential presence of low and high molecular weight ions in uninfected roots compared with nematode-induced galls. We predict the presence of lipids in both uninfected roots and galls, which was validated by MALDI time-of-flight tandem mass spectrometry and high-resolution mass spectrometry analysis of lipid extracts. Based on the isotopic ion distribution profile, both esters and glycerophospholipids were predicted compounds and may be playing an important role in gall development. Our results indicate that the MALDI-MSI technology is a promising tool to identify secondary metabolites as well as peptides and proteins in complex plant tissues like galls to decipher molecular processes responsible for infection and maintenance of these feeding sites during nematode parasitism.

Ruthenium(II) complexes of 1,3-thiazolidine-2-thione: Cytotoxicity against tumor cells and anti-Trypanosoma cruzi activity enhanced upon combination with benznidazole.

Three new mixed and mononuclear Ru(II) complexes containing 1,3-thiazolidine-2-thione (tzdtH) were synthesized and characterized by spectroscopic analysis, molar conductivity, cyclic voltammetry, high-resolution electrospray ionization mass spectra and X-ray diffraction. The complexes presented unique stereochemistry and the proposed formulae are: [Ru(tzdt)(bipy)(dppb)]PF6 (1), cis-[Ru(tzdt)2(PPh3)2] (2) and trans-[Ru(tzdt)(PPh3)2(bipy)]PF6 (3), where dppb=1,4-bis(diphenylphosphino)butane and bipy=2,2'-bipyridine. These complexes demonstrated strong cytotoxicity against cancer cell lines when compared to cisplatin. Specifically, complex 2 was the most potent cytotoxic agent against MCF-7 breast cells, while complexes 1 and 3 were more active in DU-145 prostate cells. Binding of complexes to ctDNA was determined by UV-vis titration and viscosity measurements and revealed binding constant (Kb) values in range of 1.0-4.9×10(3)M(-1), which are characteristic of compounds possessing weak affinity to ctDNA. In addition, these complexes presented antiparasitic activity against Trypanosoma cruzi. Specifically, complex 3 demonstrated strong potency, moderate selectivity index and acted in synergism with the approved antiparasitic drug, benznidazole. Additionally, complex 3 caused parasite cell death through a necrotic process. In conclusion, we demonstrated that Ru(II) complexes have powerful pharmacological activity, while the metal-free tzdtH does not provoke the same outcome.

Covalent binding and anchoring of cytochrome c to mitochondrial mimetic membranes promoted by cholesterol carboxyaldehyde.

Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.

Conformational and functional effects induced by D- and L-amino acid epimerization on a single gene encoded peptide from the skin secretion of Hypsiboas punctatus.

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy (1)H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways.

Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family.

Spider venom toxins have raised interest in prospecting new drugs and pesticides. Nevertheless, few studies are conducted with tarantula toxins, especially with species found in Brazil. This study aims to characterize chemically and biologically the first toxin isolated from Acanthoscurria paulensis venom. Ap1a consists of 48 amino acid residues and has a molecular mass of 5457.79 Da. The cloned gene encodes a putative sequence of 23 amino acid residues for the signal peptide and 27 for the pro-peptide. The sequence of the mature peptide is 60-84% identical with those of toxins of the HWTX-II family. Different from the structural pattern proposed for these toxins, the disulfide pairing of Ap1a is of the ICK type motif, which is also shared by the U1-TRTX-Bs1a toxin. Ap1a induced a dose-dependent and reversible paralytic effect in Spodoptera frugiperda caterpillars, with an ED50 of 13.0 ± 4.2 µg/g 8 h after injections. In the Drosophila melanogaster Giant Fiber circuit, Ap1a (1.14-22.82 µg/g) reduces both the amplitude and frequency of responses from GF-TTM and GF-DLM pathways, suggesting an action at the neuromuscular junction, which is mediated by glutamatergic receptors. It is also lethal to mice (1.67 µg/g, intracranial route), inducing effects similar to those reported with intracerebroventricular administration of NMDA. Ap1a (1 µM) does not alter the response induced by acetylcholine on the rhabdomyosarcoma cell preparation and shows no significant effects on hNav1.2, hNav1.4, hNav1.5, and hNav1.6 channels. Because of its unique sequence and cysteine assignment to the HWTX-II family, Ap1a is a significant contribution to the structure-function study of this family of toxins.

Peptidomic dissection of the skin secretion of Phasmahyla jandaia (Bokermann and Sazima, 1978) (Anura, Hylidae, Phyllomedusinae).

The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaço range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.

Structural and functional analyses of Mycobacterium tuberculosis Rv3315c-encoded metal-dependent homotetrameric cytidine deaminase.

The emergence of drug-resistant strains of Mycobacterium tuberculosis, the causative agent of tuberculosis, has exacerbated the treatment and control of this disease. Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that recycles cytidine and 2'-deoxycytidine for uridine and 2'-deoxyuridine synthesis, respectively. A probable M. tuberculosis CDA-coding sequence (cdd, Rv3315c) was cloned, sequenced, expressed in Escherichia coli BL21(DE3), and purified to homogeneity. Mass spectrometry, N-terminal amino acid sequencing, gel filtration chromatography, and metal analysis of M. tuberculosis CDA (MtCDA) were carried out. These results and multiple sequence alignment demonstrate that MtCDA is a homotetrameric Zn(2+)-dependent metalloenzyme. Steady-state kinetic measurements yielded the following parameters: K(m)=1004 microM and k(cat)=4.8s(-1) for cytidine, and K(m)=1059 microM and k(cat)=3.5s(-1) for 2'-deoxycytidine. The pH dependence of k(cat) and k(cat)/K(M) for cytidine indicate that protonation of a single ionizable group with apparent pK(a) value of 4.3 abolishes activity, and protonation of a group with pK(a) value of 4.7 reduces binding. MtCDA was crystallized and crystal diffracted at 2.0 A resolution. Analysis of the crystallographic structure indicated the presence of a Zn(2+) coordinated by three conserved cysteines and the structure exhibits the canonical cytidine deaminase fold.

Post-secretory events alter the peptide content of the skin secretion of Hypsiboas raniceps.

A novel family of antimicrobial peptides, named raniseptins, has been characterized from the skin secretion of the anuran Hypsiboas raniceps. Nine cDNA molecules have been successfully cloned, sequenced, and their respective polypeptides were characterized by mass spectrometry and Edman degradation. The encoded precursors share structural similarities with the dermaseptin prepropeptides from the Phyllomedusinae subfamily and the mature 28-29 residue long peptides undergo further proteolytic cleavage in the crude secretion yielding consistent fragments of 14-15 residues. The biological assays performed demonstrated that the Rsp-1 peptide has antimicrobial activity against different bacterial strains without significant lytic effect against human erythrocytes, whereas the peptide fragments generated by endoproteolysis show limited antibiotic potency. MALDI imaging mass spectrometry in situ studies have demonstrated that the mature raniseptin peptides are in fact secreted as intact molecules within a defined glandular domain of the dorsal skin, challenging the physiological role of the observed raniseptin fragments, identified only as part of the crude secretion. In this sense, stored and secreted antimicrobial peptides may confer distinct protective roles to the frog.

Cloning, molecular characterization and heterologous expression of AMY1, an alpha-amylase gene from Cryptococcus flavus.

A Cryptococcus flavus gene (AMY1) encoding an extracellular alpha-amylase has been cloned. The nucleotide sequence of the cDNA revealed an ORF of 1896 bp encoding for a 631 amino acid polypeptide with high sequence identity with a homologous protein isolated from Cryptococcus sp. S-2. The presence of four conserved signature regions, (I) (144)DVVVNH(149), (II) (235)GLRIDSLQQ(243), (III) (263)GEVFN(267), (IV) (327)FLENQD(332), placed the enzyme in the GH13 alpha-amylase family. Furthermore, sequence comparison suggests that the C. flavusalpha-amylase has a C-terminal starch-binding domain characteristic of the CBM20 family. AMY1 was successfully expressed in Saccharomyces cerevisiae. The time course of amylase secretion in S. cerevisiae resulted in a maximal extracellular amylolytic activity (3.93 U mL(-1)) at 60 h of incubation. The recombinant protein had an apparent molecular mass similar to the native enzyme (c. 67 kDa), part of which was due to N-glycosylation.

Crystallization and preliminary crystallographic studies of Schizolobium parahyba chymotrypsin inhibitor (SPCI) at 1.8 A resolution.

SPCI, a Kunitz-type chymotrypsin inhibitor, is a 180-amino-acid polypeptide isolated from Schizolobium parahyba seeds. This inhibitor has been characterized as a highly stable protein over a broad pH and temperature range. SPCI was crystallized using a solution containing 0.1 M sodium acetate trihydrate buffer pH 4.6, 33%(v/v) PEG 2000 and 0.2 M ammonium sulfate. Data were collected to 1.80 A resolution from a single crystal of SPCI under cryogenic conditions. The protein crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 40.01, b = 71.58, c = 108.68 A and an R(merge) of 0.052. The structure of SPCI has been solved by molecular replacement using the known structure of the Kunitz-type trypsin inhibitor from Delonix regia (PDB code 1r8n) as the search model.

Crystallization of Ranasmurfin, a blue-coloured protein from Polypedates leucomystax.

Ranasmurfin, a previously uncharacterized approximately 13 kDa blue protein found in the nests of the frog Polypedates leucomystax, has been purified and crystallized. The crystals are an intense blue colour and diffract to 1.51 A with P2(1) symmetry and unit-cell parameters a = 40.9, b = 59.9, c = 45.0 A, beta = 93.3 degrees . Self-rotation function analysis indicates the presence of a dimer in the asymmetric unit. Biochemical data suggest that the blue colour of the protein is related to dimer formation. Sequence data for the protein are incomplete, but thus far have identified no model for molecular replacement. A fluorescence scan shows a peak at 9.676 keV, indicating that the protein binds zinc and suggesting a route for structure solution.