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Significant Homology (significant + homology)
Selected AbstractsTranscriptomic study of apricot fruit (Prunus armeniaca) ripening among 13 006 expressed sequence tagsPHYSIOLOGIA PLANTARUM, Issue 3 2005Jérôme Grimplet To improve the knowledge of fruit ripening and to provide genomic resources for molecular breeding of apricot (Prunus armeniaca L), 13 006 expressed sequence tags (ESTs) were generated from three ,zap cDNA libraries of the pericarp tissues at different stages of development (Physiol Plant 105: 294,303), yielding 5219 (40%) Unigenes. At this stage, the very low interlibrary redundancy indicated that EST sampling of the transcriptome of apricot pericarp is still far from being saturated. Seventy-six percent of Unigenes displayed homologies with public sequences and were clustered into functional categories. The largest expressed categories were related to primary metabolism, stress response, and protein synthesis. Electronic Northern analysis revealed that stress-related proteins and cell wall modification-related enzymes strongly increased during ripening. Among 448 isoproteins (amino acid-level isogenes) detected in the Unigene set, 186 (42%) displayed significant homologies in their coding regions (nucleic acid-level isogenes). [source] Protein tyrosine phosphatases in Chaetopterus egg activationDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5-6 2003Shantá D. Hinton Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTP,, -,, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with ,-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process. [source] Novel DNA repair alkyltransferase from Caenorhabditis elegansENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 2-3 2001Sreenivas Kanugula Abstract O6 -Alkylguanine DNA-alkyltransferase (AGT) is a widely distributed DNA repair protein that protects living organisms from endogenous and exogenous alkylation damage to DNA at the O6 -position of guanine. The search of the C. elegans genome database for an AGT protein revealed the presence of a protein (cAGT-2) with some similarity to known AGTs in addition to the easily recognized cAGT-1 protein. The predicted protein sequence of cAGT-2 contains the amino acid sequence ,ProCysHisPro, at the presumed active site of the protein, whereas all other known AGTs have ,ProCysHisArg,. A truncated version of the cAGT-2 protein was expressed in E. coli. This purified recombinant protein was able to repair O6 -methylguanine and O4 -methylthymine adducts in DNA in vitro and also reacted with the bulky benzyl adduct in O6 -benzylguanine. This fragment of cAGT-2 (104 amino acids) is the smallest protein possessing AGT activity yet described. The full-length cAGT-2 protein (274 amino acids) totally lacks the N-terminal domain present in all other known AGTs but has a long C-terminal extension that has significant homology to histone 1C. Expression of cAGT-2 in an E. coli strain lacking endogenous AGT activity provided modest but statistically significant resistance to the toxicity of N -methyl- N,-nitro- N -nitrosoguanidine, confirming that cAGT-2 is an alkyltransferase. Environ. Mol. Mutagen. 38:235,243, 2001. © 2001 Wiley-Liss, Inc. [source] Identification of tudor repeat associator with PCTAIRE 2 (Trap)FEBS JOURNAL, Issue 7 2000A novel protein that interacts with the N-terminal domain of PCTAIRE 2 in rat brain PCTAIRE 2 is a Cdc2-related kinase that is predominantly expressed in the terminally differentiated neuron. To elucidate the function of PCTAIRE 2, proteins that associate with PCTAIRE 2 were screened by the yeast two-hybrid system. A positive clone was found to encode a novel protein that could bind to PCTAIRE 2 in vitro as well as in vivo, and was designated as Trap (tudor repeat associator with PCTAIRE 2). The overall structure of Trap shows no significant homology to any proteins, but contains five repeated domains (the tudor-like domain), conserved in Drosophila tudor protein. Trap associates with the N-terminal domain of PCTAIRE 2 through its C-terminal domain, which contains two tudor-like domains. PCTAIRE 1, but not PCTAIRE 3, can also associate with Trap. Trap is predominantly expressed in brain and testis, and gradually increases during brain development throughout life, consistent with the expression pattern of PCTAIRE 2. Immunoreactivities for PCTAIRE 2 and Trap were colocalized to the mitochondria in COS 7 cells. Immunohistochemical analyses showed that PCTAIRE 2 and Trap were distributed in the same cell layer of the cerebral cortex and cerebellum. These findings suggest that Trap is a physiological partner of PCTAIRE 2 in terminally differentiated neurons. [source] Subcellular localization of proteins labeled with GFP in Xanthomonas citri ssp. citri: targeting the division septumFEMS MICROBIOLOGY LETTERS, Issue 1 2010Paula M.M. Martins Abstract Xanthomonas citri ssp. citri (Xac) is the causal agent of citrus canker, an economically important disease that affects citrus worldwide. To initiate the characterization of essential biological processes of Xac, we constructed integrative plasmids for the ectopic expression of green fluorescent protein (GFP)-labeled proteins within this bacterium. Here, we show that the disruption of the ,-amylase gene (amy), the site of plasmid integration into the bacterial chromosome, does not alter its pathogenesis while abolishing completely the ability of Xac to degrade starch. Furthermore, our GFP expression system was used to characterize ORF XAC3408, a hypothetical protein encoded by Xac that shares significant homology to the FtsZ-stabilizing factor ZapA from Bacillus subtilis (ZapABsu). GFP-XAC3408 expressed in Xac exhibited a septal localization pattern typical of GFP-ZapABsu, which indicates that XAC3408 is the Xac orthologue of the cell division protein ZapABsu. The results demonstrate the potential of GFP labeling for protein functional characterizations in Xac, and, in addition, the Xac mutant strain labeled at the septum constitutes a biological model for the exploration of antibacterial compounds able to inhibit cell division in this plant pathogen. [source] Purification and characterization of a glutathione S -transferase from the fungus Cunninghamella elegansFEMS MICROBIOLOGY LETTERS, Issue 2 2001Chang-Jun Cha Abstract Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S -transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120,000×g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate,polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of Mr 27,000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian ,-, ,-, and ,-class GSTs, although it showed a small degree of cross-reactivity with a ,-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs. [source] The ancestral complement system in sea urchinsIMMUNOLOGICAL REVIEWS, Issue 1 2001L. Courtney Smith Summary: The origin of adaptive immunity in the vertebrates can be traced to the appearance of the ancestral RAG genes in the ancestral jawed vertebrate; however, the innate immune system is more ancient. A central subsystem within innate immunity is the complement system, which has been identified throughout and seems to be restricted to the deuterostomes. The evolutionary history of complement can be traced from the sea urchins (members of the echinoderm phylum), which have a simplified system homologous to the alternative pathway, through the agnathans (hagfish and lamprey) and the elasmobranchs (sharks and rays) to the teleosts (bony fish) and tetrapods, with increases in the numbers of complement components and duplications in complement pathways. Increasing complexity in the complement system parallels increasing complexity in the deuterostome animals. This review focuses on the simplest of the complement systems that is present in the sea urchin. Two components have been identified that show significant homology to vertebrate C3 and factor B (Bf), called SpC3 and SpBf, respectively. Sequence analysis from both molecules reveals their ancestral characteristics. Immune challenge of sea urchins indicates that SpC3 is inducible and is present in coelomic fluid (the body fluids) in relatively high concentrations, while SpBf expression is constitutive and is present in much lower concentrations. Opsonization of foreign cells and particles followed by augmented uptake by phagocytic coelomocytes appears to be a central function for this simpler complement system and important for host defense in the sea urchin. These activities are similar to some of the functions of the homologous proteins in the vertebrate complement system. The selective advantage for the ancestral deuterostome may have been the amplification feedback loop that is still of central importance in the alternative pathway of complement in higher vertebrates. Feedback loop functions would quickly coat pathogens with complement leading to phagocytosis and removal of foreign cells, a system that would be significantly more effective than an opsonin that binds upon contact as a result of simple diffusion. An understanding of the immune response of the sea urchin, an animal that is a good estimator of what the ancestral deuterostome immune system was like, will aid us in understanding how adaptive immunity might have been selected for during the early evolution of the vertebrates and how it might have been integrated into the pre-existing innate immune system that was already in place in those animals. The authors are grateful to Drs Sham Nair and Paul Gross for their critique of the manuscript and helpful suggestions. This work was supported by the National Science Foundation (MCB 9603086). [source] Identification and prevalence of an enterotoxin-related gene, se-int, in Staphylococcus intermedius isolates from dogs and pigeonsJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2004K. Futagawa-Saito Abstract Aims:, To determine the prevalence of enterotoxin-producing Staphylococcus intermedius in dogs and pigeons. Methods and Results:, A total of 106 S. intermedius isolates from 44 dogs and 62 pigeons were tested for the production of enterotoxins A, B, C and D by reverse passive latex agglutination (RPLA) and for sec-canine by PCR. Only one isolate from dog was positive for SEC and sec-canine. Screening of sec-canine -negative strains by nested PCR led to the identification of a novel enterotoxin-related gene, se-int. SE-int showed a significant homology (59,61% identity) with SEC and (56·6% identity) SEB. All 44 isolates from dogs and five isolates (8·1%) from pigeons were se-int positive. Conclusions:, While S. intermedius was isolated more frequently from pigeons than from dogs, se-int was more prevalent among the S. intermedius isolates from dogs, compared with the pigeon isolates. Significance and Impact of the Study:, Further characterization of the se-int -positive S. intermedius strains should clarify their pathogenic potential including enterotoxigenicity and zoonotic transmissibility to human beings. [source] Identification of a novel protein from glial cells based on its ability to interact with NF-,B subunitsrJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2003Thersa Sweet Abstract Nuclear factor ,B (NF-,B) represents a family of inducible DNA-binding transcription factors whose activity is critical for expression of the HIV-1 genome in a broad range of cells. In addition to its interaction with the ,B DNA sequence, the association of NF-,B subunits with other cellular proteins plays an important role in stimulation of HIV-1 gene transcription in astrocytic cells. Here, we utilized a yeast two-hybrid system to screen a cDNA library from a human astrocytic cell line and were able to isolate a partial cDNA belonging to a gene with an open reading frame of 1,871 amino acid residues which binds to both the p50 and p65 subunits of NF-,B. This gene, named NF-,B-binding protein (NFBP) is located on chromosome 10q24.2-25.1 and hybridized to a single transcript of nearly 6 kb in size. It is localized to the nucleus, specifically the nucleolus of cells. Extensive computer analysis was performed with the sequence of the full length NFBP and significant homology was found between NFBP, and yeast and mouse proteins. A discussion of the potential roles of NFBP in normal and viral infected cells is included. © 2003 Wiley-Liss, Inc. [source] Novel putative nonprotein-coding RNA gene from 11q14 displays decreased expression in brains of patients with schizophreniaJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2003Oxana O. Polesskaya Abstract A modified method of differential display was employed to identify a novel gene (named PSZA11q14), the expression of which was reduced in brains from patients with schizophrenia. Decreased expression of PSZA11q14 was identified initially in Brodmann's area (BA) 21 from a small group of patients with schizophrenia (n = 4) and normal controls (n = 6) and was confirmed subsequently using independent RT-PCR assay in BA 21, 22, and 9, and in hippocampus from a larger group of patients with schizophrenia (n = 36) and controls (n = 35). PSZA11q14 is located on chromosome 11q14, an area shown previously to co-segregate with schizophrenia and related disorders in several families. Decreased expression of PSZA11q14 in patients with schizophrenia and its location on 11q14 provide converging lines of evidence indicating that PSZA11q14 may be involved in at least some cases of schizophrenia. PSZA11q14 shows no significant homology with any known gene. It has no introns and produces two RNA transcripts of ,4.5 and ,7.0 kb. The largest open reading frame (ORF) in the PSZA11q14 transcripts may potentially encode for a short polypeptide of 71 amino acids. High frequency of rare codons, the short size of this ORF, and low homology with mouse sequences, however, indicate that PSZA11q14 may instead represent a novel member of a family of nonprotein-coding RNA genes that are not translated and that function at the RNA level. PSZA11q14 is located within the first intron of the DLG-2 gene and transcribed in the opposite direction to DLG-2. These results suggest that PSZA11q14 may be considered a candidate gene for schizophrenia acting as an antisense regulator of DLG-2, which controls assembling functional N -methyl- D -aspartate (NMDA) receptors. © 2003 Wiley-Liss, Inc. [source] Microsatellite markers in peach [Prunus persica (L.) Batsch] derived from an enriched genomic and cDNA librariesMOLECULAR ECOLOGY RESOURCES, Issue 3 2002T. Yamamoto Abstract Twenty-four and 12 microsatellite loci were developed in peach [Prunus persica (L) Batsch cv. Akatsuki] by using an enriched genomic and fruit cDNA libraries, respectively. The microsatellite loci obtained from an enriched library produced 1,9 alleles per locus, 24 in total, of which 22 showed polymorphisms. The average values of observed and expected heterozygosities among the 24 loci were 0.15 and 0.68, respectively. The microsatellite loci derived from cDNA showed 1,7 alleles per locus. Eight sequences showed significant homology to the registered genes in a database. [source] Haem utilization in Vibrio cholerae involves multiple TonB-dependent haem receptorsMOLECULAR MICROBIOLOGY, Issue 3 2001Alexandra R. Mey Vibrio cholerae has multiple iron transport systems, one of which involves haem uptake through the outer membrane receptor HutA. A hutA mutant had only a slight defect in growth using haemin as the iron source, and we show here that V. cholerae encodes two additional TonB-dependent haem receptors, HutR and HasR. HutR has significant homology to HutA as well as to other outer membrane haem receptors. Membrane fractionation confirmed that HutR is present in the outer membrane. The hutR gene was co-transcribed with the upstream gene ptrB, and expression from the ptrB promoter was negatively regulated by iron. A hutA, hutR mutant was significantly impaired, but not completely defective, in the ability to use haemin as the sole iron source. HasR is most similar to the haemophore-utilizing haem receptors from Pseudomonas aeruginosa and Serratia marcescens. A mutant defective in all three haem receptors was unable to use haemin as an iron source. HutA and HutR functioned with either V. cholerae TonB1 or TonB2, but haemin transport through either receptor was more efficient in strains carrying the tonB1 system genes. In contrast, haemin uptake through HasR was TonB2 dependent. Efficient utilization of haemoglobin as an iron source required HutA and TonB1. The triple haem receptor mutant exhibited no defect in its ability to compete with its Vib, parental strain in an infant mouse model of infection, indicating that additional iron sources are present in vivo. V. cholerae used haem derived from marine invertebrate haemoglobins, suggesting that haem may be available to V. cholerae growing in the marine environment. [source] Izumo is part of a multiprotein family whose members form large complexes on mammalian spermMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 12 2009Diego A. Ellerman Izumo, a sperm membrane protein, is essential for gamete fusion in the mouse. It has an Immunoglobulin (Ig) domain and an N-terminal domain for which neither the functions nor homologous sequences are known. In the present work we identified three novel proteins showing an N-terminal domain with significant homology to the N-terminal domain of Izumo. We named this region "Izumo domain," and the novel proteins "Izumo 2," "Izumo 3," and "Izumo 4," retaining "Izumo 1" for the first described member of the family. Izumo 1,3 are transmembrane proteins expressed specifically in the testis, and Izumo 4 is a soluble protein expressed in the testis and in other tissues. Electrophoresis under mildly denaturing conditions, followed by Western blot analysis, showed that Izumo 1, 3, and 4 formed protein complexes on sperm, Izumo 1 forming several larger complexes and Izumo 3 and 4 forming a single larger complex. Studies using different recombinant Izumo constructs suggested the Izumo domain possesses the ability to form dimers, whereas the transmembrane domain or the cytoplasmic domain or both of Izumo 1 are required for the formation of multimers of higher order. Co-immunoprecipitation studies showed the presence of other sperm proteins associated with Izumo 1, suggesting Izumo 1 forms a multiprotein membrane complex. Our results raise the possibility that Izumo 1 might be involved in organizing or stabilizing a multiprotein complex essential for the function of the membrane fusion machinery. Mol. Reprod. Dev. 76: 1188,1199, 2009. © 2009 Wiley-Liss, Inc. [source] Cloning of nodule-specific cDNAs of Galega orientalisPHYSIOLOGIA PLANTARUM, Issue 4 2002Seppo Kaijalainen Differential display was applied in order to clone cDNAs expressed exclusively or predominantly in nodules, compared to uninoculated root tissue of Galega orientalis. Forty-five fragments were unique for nodule RNA. These fragments were reamplified and cloned. Six of them produced a nodule-specific signal on Northern hybridization. These six fragments were sequenced. Five of the sequenced fragments showed homology to nodulin-gene sequences in databases, among them Vicia faba mRNA for protein showing partial homology with Medicago sativa nodulin-25 (Nms25), Pisum sativum PsN466, V. faba CCP2 and CCP4, P. sativum ENOD3, and Maackia amurensis ENOD2. The remaining sequence had no significant homology with sequences in the databanks. Full-size cDNA for the homologue to V. faba mRNA for the protein showing partial homology with M. sativa nodulin-25 (Nms25) and P. sativum PsN466 were cloned and sequenced. [source] Structural-based mutational analysis of d -aminoacylase from Alcaligenes faecalis DA1PROTEIN SCIENCE, Issue 11 2002Cheng-Sheng Hsu Abstract d -Aminoacylase is an attractive candidate for commercial production of d -amino acids through its catalysis in the zinc-assistant hydrolysis of N -acyl- d -amino acids. We report here the cloning, expression, and structural-based mutation of the d -aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue d -aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel ,/,-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in d -aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization. [source] Characterization of a Cryptosporidium parvum Gene Encoding a Protein with Homology to Long Chain Fatty Acid SynthetaseTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2003Leonardo Camero ABSTRACT: We describe here the cloning, sequencing, and characterization of a novel Cryptosporidium parvum gene, encoding a protein with significant homology to the long-chain fatty acyl-CoA synthetase (LCFA, EC 6.2.13). The gene has an open reading frame of 2,301 bp, coding for a 766 amino acid polypeptide, and with an estimated MW of 86.1 kDa. By indirect immunofluorescence assay, monoclonal antibodies C3CE7 and ESD labeled the anterior pole of fixed C. parvum sporozoites and developmental stages in C. parvum-infected cultures at 24, 48, and 72 h post-infection. These monoclonal antibodies inhibited more than 3.5% of parasite growth in vitro. The effect of triacsin C, a potent selective inhibitor of LCFA synthetase, on parasite growth was assessed in cell culture; complete inhibition of parasite growth at 2.5 ug/inl was obtained with little evidence of drug-associated cytotoxicity. These results suggest that the fatty acyl-CoA synthetase may be a useful target in the development of selective inhibitors and immunologic interventions against C. parvum [source] Linkage mapping and comparative analysis of bovine expressed sequence tags (ESTs)ANIMAL GENETICS, Issue 3 2000W M Grosse Summary Bovine expressed sequence tags (ESTs) containing microsatellites are suitable markers for both linkage and comparative maps. We isolated clones from a bovine fetal thigh skeletal muscle cDNA library that were positive for a (CA)10 probe. Thirty individual clones were isolated and characterised by sequencing. Sequences from the 5, and 3, ends of a clone were considered as separate ESTs until a contiguous sequence was identified. A total of 47 ESTs were sequenced from the 5, and/or 3, ends and full sequence was obtained for the 30 clones. BLAST nucleotide analysis identified significant homology to known mammalian coding regions for 31 of the bovine ESTs, 30 of which also matched human ESTs or sequence-tagged sites (STS). The remaining 16 bovine ESTs represented novel transcripts. Microsatellites were isolated in 27 of the ESTs, 11 of which were developed into markers and placed on the MARC bovine linkage map. Human cytogenetic map positions were available for 20 of the 30 human EST orthologs, and a putative bovine map position for 17 of the sequences could be inferred using comparative mapping data. These results demonstrated that mapping bovine ESTs containing microsatellites is a plausible strategy to increase the density of gene markers on the bovine linkage and comparative maps. [source] Identifications of expressed sequence tags from Pacific threadfin (Polydactylus sexfilis) skeletal muscle cDNA libraryAQUACULTURE RESEARCH, Issue 4 2010Shizu Watanabe Abstract Pacific threadfin (Polydactylus sexfilis), locally known as Moi, is a desirable fish for aquaculture and recreational fishing. To understand the basic mechanism of muscle formation and its impacts on flesh quality, we established a cDNA library using mRNA of the skeletal muscle tissue from fingerlings. The library size was 1.1 × 108 plaque forming units mg,1 and the percentage of recombinant clones was >81%. A pilot sequencing project from 181 clones identified 129 useful expressed sequence tags (ESTs), of which 90 ESTs exhibited significant homology to known genes and 39 ESTs have low homologies to unknown genes by blast algorithm. The most abundant EST from the pilot sequence data is nikotinamide riboside kinase 2 (59 times), followed by 60S ribosomal protein L24 (12 times) and ribosomal protein L8 (5 times). Fourteen novel genes were retrieved from the sequenced clones and subjected to gene ontology annotation. Four mRNA sequences were identified as significant regulators of transcription, including Not2p, Tsc22 domain family 2, LIM domain binding factor 3 and mesenchyme homeobox 2. These results suggest that the muscle cDNA library is an useful source for identifying EST sequences of Pacific threadfin. [source] Investigation of de novo Totally Random Biosequences, Part IICHEMISTRY & BIODIVERSITY, Issue 8 2006On the Folding Frequency in a Totally Random Library of de novo Proteins Obtained by Phage Display Abstract We present an investigation on theoretically possible protein structures which have not been selected by evolution and are, therefore, not present on our Earth (,Never Born Proteins' (NBP)). In particular, we attempt to assess whether and to what extent such polypeptides might be folded, thus acquiring a globular protein status. A library (ca. 109 clones) of totally random polypeptides, with a length of 50 amino acids, has been produced by phage display. The only structural bias in these sequences is a tripeptide substrate for thrombin: PRG, chosen according to the criteria described in the preceding Part,I of this series. The presence of this substrate in an otherwise totally random sequence forms the basis for a qualitative experimental criterion which distinguishes unfolded from folded proteins, as folded proteins are more protected from protease digestion than unfolded ones. The investigation of 79 sequences, randomly selected from the initially large library, shows that over 20% of this population is thrombin-resistant, likely due to folding. Analysis of the amino acid sequences of these clones shows no significant homology to extant proteins, which indicates that they are indeed totally de novo. A few of these sequences have been expressed, and here we describe the structural properties of two thrombin-resistant randomly selected ones. These two de novo proteins have been characterized by spectroscopic methods and, in particular, by circular dichroism. The data show a stable three-dimensional folding, which is temperature-resistant and can be reversibly denatured by urea. The consequences of this finding within a library of ,Never Born Proteins' are discussed in terms of molecular evolution. [source] | |||||||||||||