Electron-paramagnetic-resonance spectroscopy of iron-binding fragments of hen ovotransferrins.

1. It is confirmed that there are two e.p.r. (electron-paramagnetic-resonance) signals associated with fully loaded ovotransferrin, which has two iron-binding sites. 2. Through experiments in which either of the two sites of whole ovotransferrin is occupied, the other being empty, the first occupied site is shown to belong to the N-terminal region of the protein; the second occupied site is in the C-terminal region. 3. When the protein is cleaved with trypsin or subtilisin, the N-terminal fragments are spectroscopically similar to the monoferric ovotransferrin complexes in which the iron atom occupies the N-terminal or C-terminal site respectively. Each fragment displays the same two e.p.r. signals, though not in the same proportions. 4. Computer summations of the e.p.r. spectra confirm that there is no iron-iron interaction which affects the spin Hamiltonian parameters at the iron-binding sites.     

The dimerization of half-molecule fragments of transferrin.

Partial proteolysis was used to prepare half-molecule fragments of hen ovotransferrin. N-Terminal and C-terminal fragments associate to form an N-terminal fragment-C-terminal fragment dimer. Variant forms of the N- and C-terminal fragments can be prepared in which a few amino acid residues are lacking from the C-terminal ends of the fragments. These variant fragments are partially or completely unable to associate; the suggestion that the molecular recognition sites are located in these C-terminal stretches of the N-terminal half-molecule (320-332) and of the C-terminal half-molecule (683-686) is in agreement with X-ray-crystallography data for human lactotransferrin [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1769-1773].     

The formation of iron-binding fragments of hen ovotransferrin by limited proteolysis

1. Iron was added to hen ovotransferrin to 30% saturation and the protein was digested with trypsin or chymotrypsin. 2. Iron-binding fragments were isolated. They carried one atom of iron/mol (mol.wt. 35000) and consisted of a single polypeptide chain derived from the N-terminal half of the protein. Carbohydrate was not present. 3. The fragments were able to bind a variety of metals and to donate iron to reticulocytes.     

An extended-X-ray-absorption-fine-structure investigation of diferric transferrins and their iron-binding fragments.

Iron K-edge extended-X-ray-absorption-fine-structure (e.x.a.f.s.) spectra were recorded for diferric human and rabbit serum transferrins and for diferric chicken ovotransferrin in aqueous solution; for ovotransferrin e.x.a.f.s. spectra from the N-terminal and C-terminal domain fragments were also measured. The overall spectral profiles closely resemble one another, indicating similar iron-binding sites. The simulation of the diferric ovotransferrin spectrum suggests a first co-ordination shell consisting of six low-Z ligands (nitrogen/oxygen), two ligands at a distance of approx. 0.185 nm (1.85 A) and four ligands at approx. 0.204 nm (2.04 A). The two shorter distances may correspond to Fe-O (tyrosine), whereas the longer distance is consistent with Fe-N (histidine) and Fe-O (water). Detailed analysis of the spectra of the N-terminal and C-terminal fragments indicates a difference in the short ligand distance.     

End-label fingerprintings show that the N- and C-termini of actin are in the contact site with gelsolin

Gelsolin was cleaved by chymotrypsin or thermolysin into an N-terminal Mr 45,000 fragment (45N) and a C-terminal Mr 38,000 fragment (38C). The N-terminal half was further cleaved into two fragments with Mr 17,000 (17N) and Mr 28,000 (28N). These fragments were complexed with actin and cross-linked with 1-ethyl-3-[3-(dimethylamino)prophyl]carbodiimide (EDC) to introduce covalent bonds into their contact sites. The location of these bonds was mapped along the actin sequence by end-label fingerprinting with highly sensitive probes for the N- and C-termini of actin. The mapping studies revealed that two gelsolin N-terminal fragments (17N and 28N) were cross-linked with the actin C-terminal segment. The result indicates that the actin N- and C-terminal segments are in the binding site of gelsolin.     

Perturbed angular correlation studies of the metal-binding sites in ovotransferrin and its C- and N-terminal halves

The perturbed angular correlation (PAC) technique has been applied to study the electric quadrupole interaction of ¹⁸¹Hf nuclei at the binding sites of ovotransferrin (OTF) molecules. Two specific electric field gradients were observed. Their relative intensities depend on the pH value and the temperature of the samples, whereas the electric quadrupole interaction parameters themselves remain unaffected. In order to compare the binding sites in OTF, experiments with N- and C-terminal half-molecules were performed. Both specific configurations are observed at the N-terminal and at the C-terminal binding site with similar quadrupole parameters as for the intact protein. Remarkably, the stability of the hafnium binding to the C-terminal fragment appears to be reduced as compared with the N-terminal half and the intact protein.     

The iron-binding properties of hen ovotransferrin.

1. The distribution of iron between the two iron-binding sites in partially saturated ovotransferrin was studied by labelling with 55Fe and 59Fe and by gel electrophoresis in a urea-containing buffer. 2. When iron is added in the form of chelate complexes at alkaline pH, binding occurs preferentially at the N-terminal binding site. In acid, binding occurs preferentially at the C-terminal site. 3. When simple iron donors (ferric and ferrous salts) are used the metal is distributed at random between the binding sites, as judged by the gel-electrophoresis method. The double-isotope method shows a preference of ferrous salts for the N-terminal site. 4. Quantitative treatment of the results of double-isotope labelling suggests that in the binding of iron to ovotransferrin at alkaline pH co-operative interactions between the sites occur. These interactions are apparently absent in the displacement of copper and in the binding of iron at acid pH.     

Intrachain disulphide bridges in immunoglobulin G heavy chains. The Fc fragment

The disulphide bridges of the Fc fragment (C-terminal half of the heavy chain) have been studied in several human immunoglobulins, containing heavy chains of different antigenic types (gamma1, gamma2, gamma3 and gamma4), and in heavy-chain-disease proteins. Two intrachain disulphide bridges were found to be present. The sequences appear to be identical in the Fc fragments of two types of chain studied (gamma1 and gamma3), and very similar to corresponding sequences of the Fc fragment in rabbit. These results suggest that the C-terminal half of the heavy chains is covalently folded (in a similar fashion to the light chains) with a C-terminal loop and an N-terminal loop. The similarity is emphasized by comparison of the sequence and location of the disulphide-bridged peptides of the C-terminal loop of heavy and light chains. The N-terminal loop, on the other hand, appears to be very different in Fc fragments and light chains. The C-terminal loop is the only one present in the F'c fragment.     

Studies of the binding of different iron donors to human serum transferrin and isolation of iron-binding fragments from the N- and C-terminal regions of the protein.

1. Trypsin digestion of human serum transferrin partially saturated with iron(III)-nitrilotriacetate at pH 5.5 or pH 8.5 produces a carbohydrate-containing iron-binding fragment of mol.wt. 43000. 2. When iron(III) citrate, FeCl3, iron (III) ascorabate and (NH4)2SO4,FeSO4 are used as iron donors to saturate the protein partially, at pH8.5, proteolytic digestion yields a fragment of mol.wt. 36000 that lacks carbohydrate. 3. The two fragments differ in their antigenic structures, amino acid compositions and peptide 'maps'. 4. The fragment with mol.wt. 36000 was assigned to the N-terminal region of the protein and the other to the C-terminal region. 5. The distribution of iron in human serum transferrin partially saturated with various iron donors was examined by electrophoresis in urea/polyacrylamide gels and the two possible monoferric forms were unequivocally identified. 6. The site designated A on human serum transferrin [Harris (1977) Biochemistry 16, 560--564] was assigned to the C-terminal region of the protein and the B site to the N-terminal region. 7. The distribution of iron on transferrin in human plasma was determined.     

Organization of the coiled-coils in the wool microfibril

Two helix-enriched fragments were isolated from a partial tryptic digest of the microfibrillar proteins of wool. From the known sequences of two of the constituent microfibrillar polypeptides, components 7c and 8c-1, one of these fragments was identified as arising from the N-terminal half of the helix-rich region and the other from the C-terminal half of this region. On the basis of the molecular weight in benign and dissociating solvents, crosslinking with dimethylsuberimidate, and amino acid and sequence analyses the N-terminal fragment was shown to be a tetrameric structure, consisting of two segments from each of the components 7 and 8 families. The C-terminal fragment was shown to be a two-stranded coiled-coil containing one chain segment from each of components 7 and 8. The evidence is not compatible with anything but a two-stranded coiled-coil as the basic molecular structure for the wool microfibril and provides experimental proof of a parallel arrangement of the chains. The thermal stabilities of the two fragments and of the parent microfibrillar complex from which they are derived are very similar indicating that the non-helical end regions of the polypeptide chains do not greatly affect the stability of the α-helical rod domain.     

Homotypic fibrillin-1 interactions in microfibril assembly

We have defined the homotypic interactions of fibrillin-1 to obtain new insights into microfibril assembly. Dose-dependent saturable high affinity binding was demonstrated between N-terminal fragments, between furin processed C-terminal fragments, and between these N- and C-terminal fragments. The N terminus also interacted with a downstream fragment. A post-furin cleavage site C-terminal sequence also interacted with the N terminus, with itself and with the furin-processed fragment. No other homotypic fibrillin-1 interactions were detected. Some terminal homotypic interactions were inhibited by other terminal sequences, and were strongly calcium-dependent. Treatment of an N-terminal fragment with N-ethylmaleimide reduced homotypic binding. Microfibril-associated glycoprotein-1 inhibited N- to C-terminal interactions but not homotypic N-terminal interactions. These fibrillin-1 interactions are likely to regulate pericellular fibrillin-1 microfibril assembly.     

Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies to either domain of ovotransferrin block binding to transferrin receptors on chick reticulocytes

Monoclonal antibodies produced to both chicken ovotransferrin and to the isolated N- and C-terminal half-molecule domains of ovotransferrin have been used to probe the interaction of ovotransferrin with its specific receptor on chick embryo red blood cells. Two antibodies to epitopes on the N-terminal domain and one antibody to an epitope on the C-terminal domain were able to block the binding of 125I-labeled diferric ovotransferrin to the receptor. When the cellular surface receptors were first saturated with ovotransferrin at 0 degrees C, none of these antibodies bound to the cell-associated ovotransferrin. This suggests that the antibodies are to epitopes which lie very near to, or in the regions of, the two domains which interact with receptor. The same three antibodies also blocked the binding to the receptor of ovotransferrin associated in situ from the isolated N- and C-terminal half-molecule domains. A fourth antibody did not block binding to receptor of 125I-labeled diferric ovotransferrin or the associated domains; furthermore, it was able to bind to ovotransferrin bound to the cell surface at 0 degrees C. This antibody thus appears to recognize an epitope remote from the receptor binding region of ovotransferrin. Additional evidence for the requirement of the presence of both domains of ovotransferrin to effect binding to the transferrin receptor on chick reticulocytes was obtained with a fifth antibody which recognized only the N-terminal half-molecule domain but not holo-ovotransferrin. Although this antibody had no effect on the binding of 125I-labeled ovotransferrin to cells, it blocked binding to receptor of the associated domains of ovotransferrin, presumably by inhibiting the association of the two domains.     

Amino-terminal and carboxyl-terminal half-molecules of ovotransferrin: preparation by a novel procedure and their interactions

The amino- (N-) and carboxyl- (C-)terminal half-molecules of ovotransferrin were prepared by a novel procedure. The trypsin-nicked ovotransferrin (Ikeda et al. (1985) FEBS Lett. 182, 305-309), in which the two half-molecules interact non-covalently forming a stable dimer, was purified by gel filtration and anion-exchange column chromatography. By subsequent cation-exchange chromatography, the nicked form was distinctly separated into an equivalent amount of the N-terminal and C-terminal half-molecules. Analyses of the N-terminal and C-terminal sequences indicated that the N-terminal and C-terminal half-molecules comprised the alignments of residues 1-332 and 342-686 of ovotransferrin, respectively. Anion-exchange chromatography, gel filtration chromatography, and non-denaturing polyacrylamide gel electrophoresis revealed that the isolated half-molecules had the ability to re-associate in solution. The contents of alpha-helix and beta-sheet of the two half-molecules, as determined by circular dichroism (CD) spectra, were very similar to those of intact ovotransferrin. No prominent alteration in the secondary structure of the two half-molecules was induced by the re-association.     

Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Folding properties of functional domains of tropomodulin.

Tropomodulin (Tmod) stabilizes the actin-tropomyosin filament by capping the slow-growing end (P-end). The N- and C-terminal halves play distinct roles; the N-terminal half interacts with the N-terminal region of tropomyosin, whereas the C-terminal half interacts with actin. Our previous study (A. Kostyukova, K. Maeda, E. Yamauchi, I. Krieger, and Y. Maéda Y., 2000, Eur. J. Biochem. 267:6470-6475) suggested that the two halves are also structurally distinct from each other. We have now studied the folding properties of the two halves, by circular dichroism spectroscopy and by differential scanning calorimetry of the expressed chicken E-type tropomodulin and its large fragments. The results showed that the C-terminal half represents a single, independently folded unit that melts cooperatively through a two-state transition. In contrast, the N-terminal half lacks a definite tertiary structure in solution. The binding of N11, a fragment that corresponds to the first 91 amino acids of the tropomodulin, to tropomyosin substantially stabilized the tropomyosin. This may indicate that the flexible structure of the N-terminal half of tropomodulin in solution is required for binding to tropomyosin and that the N-terminal half acquires its tertiary structure upon binding to tropomyosin.     

Use of fragments of hirudin to investigate thrombin-hirudin interaction

Site-directed mutagenesis was used to create hirudin in which Asn52 was replaced by methionine. Cyanogen bromide cleavage at this unique methionine resulted in two fragments. These fragments have been used to study the kinetic mechanism of the inhibition of thrombin by hirudin and to identify areas of the two molecules which interact with each other. The binding of the C-terminal fragment (residues 53-65) to thrombin resulted in a decrease in the Michaelis constant for the substrate D-phenylalanylpipecolylarginyl-p-nitroanilide (DPhe-Pip-Arg-NH-Ph). The N-terminal fragment (residues 1-52) was a competitive inhibitor of thrombin. There was a small amount of cooperativity in the binding of the two fragments. Whereas hirudin and its C-terminal fragment protected alpha-thrombin against cleavage by trypsin, the N-terminal fragment did not. Hirudin and the N-terminal fragment completely prevented the cleavage of alpha-thrombin by pancreatic elastase while the C-terminal fragment afforded a lesser degree of protection. The results of these experiments with trypsin and elastase are discussed in terms of interaction areas on thrombin and hirudin.     

The location of structural difference between ovotransferrin types A and B in hens

A comparison of ovotransferrin types A and B showed that in starch gel electrophoresis both types consisted of one major and one minor component. Both types have a similar amino acid composition as do the fragments from each type. Starch gel electrophoresis shows that the cause of the difference in the elec-trophoretic mobilities between ovotransferrin types A and B lies in the C-termi-nal half of the molecule. No physiological difference was found between types A and B, both types donate iron to chicken embryo red cells at equal rate.     

The carboxyl-terminal half-molecule of ovotransferrin prepared by selective digestion of the amino-terminal lobe with thermolysin.

Diferric ovotransferrin was hydrolyzed by thermolysin, a thermostable protease, at elevated temperatures. At 65 degrees C, the amino(N)-terminal lobe was completely digested into small peptides, while the carboxyl(C)-terminal lobe was significantly resistant to the protease. This permitted the isolation of an iron-bound C-terminal half-molecule consisting of a glycosylated single polypeptide in an excellent yield (about 90%). The fragment comprises the residues from 336 to the C-terminus of ovotransferrin. The results for the visible absorption spectrum of the copper-bound fragment, the stability of the iron-bound fragment in high concentration of urea, and the CD spectra of the fragment in the far and near UV regions indicated that it retains the metal binding activity and conformation of the C-terminal lobe of intact ovotransferrin.     

Unusual proteolysis of the protoxin and toxin from Bacillus thuringiensis. Structural implications

Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N- and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.