The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.     

Identification of variant molecules of Bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme A and an unexpected [3Fe-4S] cluster associated with a canonical [4Fe-4S] ligand motif

During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.     

cDNA cloning and expression of acutin

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.     

Expression, Purification, and Characterization of a [Fe2S2] Cluster Containing Ferredoxin from Acidithiobacillus ferrooxidans

The [2Fe-2S] cluster containing ferredoxin has attracted much attention in recent years. Genetic analyses show that it has an essential role in the maturation of various iron–sulfur (Fe-S) proteins and functions as a component of the complex machinery responsible for the biogenesis of Fe-S clusters. The gene of ferredoxin from A. ferrooxidans ATCC 23270 was cloned, successfully expressed in Escherichia coli, and purified by one-step affinity chromatography to homogeneity. The MALDI-TOF MS and spectra results of the recombinant protein confirmed that the iron–sulfur cluster was correctly inserted into the active site of the protein. Site-directed mutagenesis results revealed that Cys42, Cys48, Cys51, and Cys87 were ligating with the [Fe₂S₂] cluster of the protein.     

Primary structure of the gamma-subunit of the GTP-binding protein from the bovine retina

The complete amino acid sequence of the gamma-subunit of GTP-binding protein from cattle retina has been established. The polypeptide chain consists of 69 amino acid residues and contains an unusual sequence Cys35-Cys36. The molecular mass of the gamma-subunit is 8008,7.     

A selenocysteine-containing peroxiredoxin from the strictly anaerobic organism Eubacterium acidaminophilum

A strongly 75Se-labeled 22 kDa protein detected previously showed in its N-terminal sequence the highest similarity to the family of thiol-dependent peroxidases, now called peroxiredoxins. The respective gene prxU was cloned and analyzed. prxU encodes a protein of 203 amino acids (22,470 Da) and contains an in-frame UGA codon (selenocysteine) at the position of the so far strictly conserved and catalytically active Cys47. The second conserved cysteine present in 2-Cys peroxiredoxins was replaced by alanine. Heterologous expression of the Eubacterium acid-aminophilum PrxU as a recombinant selenoprotein in Escherichia coli was not possible. A cysteine-encoding mutant gene, prxU47C, containing UGC instead of UGA was strongly expressed. This recombinant PrxU47C mutant protein was purified to homogeneity by its affinity tag, but was not active as a thiol-dependent peroxidase. The identification of prxU reveals that the limited class of natural selenoproteins may in certain organisms also include isoenzymes of peroxiredoxins, previously only known as non-selenoproteins containing catalytic cysteine residues.     

Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria.

Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40-50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29-Cys38 and Cys57-Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.     

The complete amino acid sequence of echinoidin, a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina. Homologies with mammalian and insect lectins

The complete amino acid sequence of echinoidin, the proposed name for a lectin from the coelomic fluid of the sea urchin Anthocidaris crassispina, has been determined by sequencing the peptides obtained from tryptic, Staphylococcus aureus V8 protease, chymotryptic, and thermolysin digestions. Echinoidin is a multimeric protein (Giga, Y., Sutoh, K., and Ikai, A. (1985) Biochemistry 24, 4461-4467) whose subunit consists of a total of 147 amino acid residues and one carbohydrate chain attached to Ser38. The molecular weight of the polypeptide without carbohydrate was calculated to be 16,671. Each polypeptide chain contains seven half-cystines, and six of them form three disulfide bonds in the single polypeptide chain (Cys3-Cys14, Cys31-Cys141, and Cys116-Cys132), while Cys2 is involved in an interpolypeptide disulfide linkage. From secondary structure prediction by the method of Chou and Fasman (Chou, P. Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222) the protein appears to be rich in beta-sheet and beta-turn structures and poor in alpha-helical structure. The sequence of the COOH-terminal half of echinoidin is highly homologous to those of the COOH-terminal carbohydrate recognition portions of rat liver mannose-binding protein and several other hepatic lectins. This COOH-terminal region of echinoidin is also homologous to the central portion of the lectin from the flesh fly Sarcophaga peregrina. Moreover, echinoidin contains an Arg-Gly-Asp sequence which has been proposed to be a basic functional unit in cellular recognition proteins.     

Nucleotide sequence of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough)

The nucleotide sequence of the 4.7-kb SalI/EcoRI insert of plasmid pHV 15 containing the hydrogenase gene from Desulfovibrio vulgaris (Hildenborough) has been determined with the dideoxy chain-termination method. The structural gene for hydrogenase encodes a protein product of molecular mass 45820 Da. The NH2-terminal sequence of the enzyme deduced from the nucleic acid sequence corresponds exactly to the amino acid sequence determined by Edman degradation. The nucleic acid sequence indicates that a N-formylmethionine residue precedes the NH2-terminal amino acid Ser-1. There is no evidence for a leader sequence. The NH2-terminal part of the hydrogenase shows homology to the bacterial [8Fe-8S] ferredoxins. The sequence Cys-Ile-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro-Xaa-Xaa-Ala-(Ile) occurs twice both in the hydrogenase and in [8Fe-8S] ferredoxins, where the Cys residues have been shown to coordinate two [4Fe-4S] clusters [Adman, E. T., Sieker, L. C. and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996]. These results, therefore, suggest that two electron-transferring ferredoxin-like [4Fe-4S] clusters are located in the NH2-terminal segment of the hydrogenase molecule. There are ten more Cys residues but it is not clear which four of these could participate in the formation of the third cluster, which is thought to be the hydrogen binding centre. Another gene, encoding a protein of molecular mass 13493 Da, was found immediately downstream from the gene for the 46-kDa hydrogenase. The nucleic acid sequence suggests that the hydrogenase and the 13.5-kDa protein belong to a single operon and are coordinately expressed. Since dodecylsulfate gel electrophoresis of purified hydrogenase indicates the presence of a 13.5-kDa polypeptide in addition to the 46-kDa component, it is proposed that the hydrogenase from D. vulgaris (Hildenborough) is a two-subunit enzyme.     

Primary structure of the major pepsin inhibitor from the intestinal parasitic nematode Ascaris suum

The major pepsin inhibitor from Ascaris suum was isolated by affinity chromatography and chromatofocusing. Its amino acid sequence was determined by automated Edman degradation of peptide fragments. Peptides were produced by chemical and enzymatic cleavage of pyridylethylated protein and were purified by reverse-phase high-performance liquid chromatography. The inhibitor consists of 149 residues with the following sequence: QFLFSMSTGP10FICTVKDNQV20FVANLPWTML30EGDDIQVGKE40 FAARVEDCTN50VKHDMAPTCT60KPPPFCGPQD70MKMFNFVGCS80VLGNKLFIDQ90KYVRDLTAK D100 HAEVQTFREK110IAAFEEQQEN120QPPSSGMPHG130AVPAGGLSPP140PPPSFCTVQ149. It has a molecular weight of 16,396. All cysteines are engaged as disulfide bonds: Cys(13)-Cys(59), Cys(48)-Cys(66), and Cys(79)-Cys(146). The protein is probably composed of two domains connected by a short hydrophobic region. This is the first aspartyl protease inhibitor of animal origin that has been sequenced. The sequence has no significant homology with any other known protein.     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.     

A zinc finger protein from Candida albicans is involved in sucrose utilization.

A sucrose-inducible alpha-glucosidase activity that hydrolyzes sucrose in Candida albicans has been demonstrated previously. The enzyme is assayable in whole cells and was inhibited by both sucrose and maltose. A C. albicans gene (CASUC1) that affects sucrose utilization and alpha-glucosidase activity was cloned by expression in a Saccharomyces cerevisiae suc2 mutant (2102) devoid of invertase genes. CASUC1 enabled the S. cerevisiae mutant to utilize both sucrose and maltose. DNA sequence analysis revealed that CASUC1 encodes a putative zinc finger-containing protein with 28% identity to a maltose-regulatory gene (MAL63) of S. cerevisiae. The gene products of CASUC1 and MAL63 are approximately the same size (501 and 470 amino acids, respectively), and each contains a single zinc finger located at the N terminus. The zinc fingers of CASUC1 and MAL63 comprise six conserved cysteines (C6 zinc finger) and are of the general form Cys-Xaa2-Cys-Xaa6-Cys-Xaavariable-Cys-Xaa2-Cys-+ ++Xaa6-Cys (where Xaan indicates a stretch of the indicated number of any amino acids). Both contain five amino acids in the variable region. CASUC1 also complemented the maltose utilization defect of an S. cerevisiae mutant (TCY-137) containing a defined mutation in a maltose-regulatory gene. The sucrose utilization defect of type II Candida stellatoidea, a sucrase-negative mutant of C. albicans, was corrected by CASUC1. Determinations of alpha-glucosidase activity in whole cells revealed that activity was restored in transformants cultivated on either sucrose or maltose. To our knowledge, this is the first zinc finger-encoding gene, as well as the first putative regulatory gene, to be identified in C. albicans.     

Structural characterization and independent folding of a chimeric glycoprotein comprising granulocyte-macrophage colony stimulating factor and erythropoietin sequences

MEN 11300 is a hybrid glycoprotein of 297 amino acids obtained by fusion of the cDNA encoding GM-CSF with the cDNA encoding EPO followed by transfection of the hybrid gene into CHO cells. The oligonucleotide construct incorporated a spacing sequence between the two individual cDNAs which encodes eight amino acids constituting a linker peptide intended to separate the GM-CSF and EPO moieties. The recombinant MEN 11300 protein was submitted to a detailed structural characterization including the verification of the entire amino acid sequence, the assignment of the disulfide bridges pattern, the identification of the glycosylation sites and the definition of the glycosidic moiety, including site-specificity. Partial processing of the C-terminal Arg residue and the occurrence of N-glycosylation sites at Asn27, Asn155, Asn169, Asn214 were established. Moreover, O-glycosylation at Ser257 and at the N-terminal region was also detected. A large heterogeneity was observed in the N-glycans due to the presence of differently sialylated and fucosylated branched complex type oligosaccharides whereas O-linked glycans were constituted by GalGalNAc chains with a different number of sialic acids. The disulfide bridges pattern was established by direct FABMS analysis of the proteolytic digests or by ESMS analysis of HPLC purified fractions. Pairing of the eight cysteine residues resulted in Cys54-Cys96, Cys88-Cys121, Cys138-Cys292, and Cys160-Cys164. This S-S bridges pattern is identical to that occurring in the individual natural GM-CSF and EPO, thus showing that the two protein moieties in MEN 11300 can independently acquire their native three-dimensional structure.      

Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes.

Barwin is a basic protein with pI above 10 and molecular mass 13.7 kDa isolated from aqueous extracts of barley seed. The complete amino acid sequence of 125 residues has been determined by a combination of conventional protein sequencing, plasma desorption mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Three disulfide bridges have been localized as Cys31-Cys63, Cys52-Cys86, and Cys66-Cys123 both by 1H nuclear magnetic resonance spectroscopy and by plasma desorption mass spectrometry. The N-terminal residue was identified as pyroglutamate. Barwin is closely related to a peptide segment of 122 residues at the C-terminal region of the proteins encoded by two wound-induced genes in potato plants, win1 and win2, and a protein encoded by the hevein gene of rubber tree. In 77 sequence positions of 125 the barwin, win1, win2, and hevein protein sequences have amino acid sequence identity, when two gaps--one of two residues allowing for the insert of Gly23 and Ala24 and one allowing for the insert of Thr97 in the barwin sequence--are introduced in the latter three. The close sequence similarity with the proteins encoded by the wound-induced potato and rubber tree genes and the ability of the protein to bind saccharides suggest that barwin might belong to a group of proteins involved in a common defense mechanism in plants.