Fe3+ binding to ovotransferrin in the presence of alpha-amino acids.

The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe(3+)-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. 113Cd-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.     

Crystallographic and biochemical analyses of the metal-free Haemophilus influenzae Fe3+-binding protein.

The crystal structure of the iron-free (apo) form of the Haemophilus influenzae Fe(3+)-binding protein (hFbp) has been determined to 1.75 A resolution. Information from this structure complements that derived from the holo structure with respect to the delineation of the process of iron binding and release. A 21 degrees rotation separates the two structural domains when the apo form is compared with the holo conformer, indicating that upon release of iron, the protein undergoes a change in conformation by bending about the central beta-sheet hinge. A surprising finding in the apo-hFbp structure was that the ternary binding site anion, observed in the crystals as phosphate, remained bound. In solution, apo-hFbp bound phosphate with an affinity K(d) of 2.3 x 10(-3) M. The presence of this ternary binding site anion appears to arrange the C-terminal iron-binding residues conducive to complementary binding to Fe(3+), while residues in the N-terminal binding domain must undergo induced fit to accommodate the Fe(3+) ligand. These observations suggest a binding process, the first step of which is the binding of a synergistic anion such as phosphate to the C-terminal domain. Next, iron binds to the preordered half-site on the C-terminal domain. Finally, the presence of iron organizes the N-terminal half-site and closes the interdomain hinge. The use of the synergistic anion and this iron binding process results in an extremely high affinity of the Fe(3+)-binding proteins for Fe(3+) (nFbp K'(eff) = 2.4 x 10(18) M(-1)). This high-affinity ligand binding process is unique among the family of bacterial periplasmic binding proteins and has interesting implications in the mechanism of iron removal from the Fe(3+)-binding proteins during FbpABC-mediated iron transport across the cytoplasmic membrane.     

Human apolipoprotein E3 in aqueous solution. II. Properties of the amino- and carboxyl-terminal domains

Hydrodynamic, chromatographic, and spectroscopic techniques were used to study the aqueous solution properties of the two structural domains of human apolipoprotein (apo) E3. An amino-terminal thrombolytic fragment of apoE (22 kDa, residues 1-191) and a carboxyl-terminal thrombolytic fragment of apoE (10 kDa, residues 216-299) were used as models for the two domains. Sedimentation equilibrium ultracentrifugation showed that apoE and the 10-kDa model domain self-associated predominantly as tetramers. The 22-kDa model domain was primarily monomeric. Molecular weights calculated from the weight average sedimentation and diffusion coefficients or from the sedimentation coefficients and Stokes radii were in agreement with the sedimentation equilibrium results. Derived frictional coefficients suggest larger axial ratios and/or more extensive hydration for the apoE and the 10-kDa domain tetramers as compared with the 22-kDa domain. Proteolysis of apoE followed by high performance liquid chromatography showed rapid production of free 22-kDa domain, whereas the free 10-kDa domain appeared as a tetramer late in the course of the hydrolysis. Assessment by circular dichroism demonstrated that both model domains and apoE had over 54% alpha-helical content, which changed little in a detergent (octyl-beta-D-glucopyranoside) or lipid (dimyristoylphosphatidylcholine) environment. In contrast to the circular dichroism results, apoE and the 10-kDa domain showed a marked blue shift in the fluorescence maximum in a lipid environment. The results suggest that the self-association of apoE in solution as a tetramer is mediated by the carboxyl-terminal domain and that the amino- and carboxyl-terminal domains do not associate with one another. The amino-terminal domain is most likely compact and globular, whereas the carboxyl-terminal domain is probably elongated. The isolated model domains appear to have structures that are similar to those of the domains in the intact protein.     

BIAcore analysis of bovine insulin-like growth factor (IGF)-binding protein-2 identifies major IGF binding site determinants in both the amino- and carboxyl-terminal domains

In the absence of a complete tertiary structure to define the molecular basis of the high affinity binding interaction between insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs), we have investigated binding of IGFs by discrete amino-terminal domains (amino acid residues 1-93, 1-104, 1-132, and 1-185) and carboxyl-terminal domains (amino acid residues 96-279, 136-279, and 182-284) of bovine IGFBP-2 (bIGFBP-2). Both halves of bIGFBP-2 bound IGF-I and IGF-II in BIAcore studies, albeit with different affinities ((1-132)IGFBP-2, K(D) = 36.3 and 51.8 nm; (136-279)IGFBP-2HIS, K(D) = 23.8 and 16.3 nm, respectively). The amino-terminal half appears to contain components responsible for fast association. In contrast, IGF binding by the carboxyl-terminal fragment results in a more stable complex as reflected by its K(D). Furthermore, des(1-3)IGF-I and des(1-6)IGF-II exhibited reduced binding affinity to (1-279)IGFBP-2HIS, (1-132)IGFBP-2, and (136-279)IGFBP-2HIS biosensor surfaces compared with wild-type IGF. A charge reversal at positions 3 and 6 of IGF-I and IGF-II, respectively, affects binding interactions with the amino-terminal fragment and full-length bIGFBP-2 but not the carboxyl-terminal fragment.     

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway

The differential properties of anion-mediated Fe(3+) release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe(3+) release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe(3+) with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe(3+) release model in which the holo form is converted depending on the anion concentration into a "mixed ligand" intermediate that releases Fe(3+). The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 A resolution demonstrated the existence of a single bound SO(4)(2-) in the interdomain cleft, which interacts directly with Thr(461)-OG1, Tyr(431)-OH, and His(592)-NE2 and indirectly with Tyr(524)-OH. The latter three groups are Fe(3+)-coordinating ligands, strongly suggesting the facilitated Fe(3+) release upon the anion occupation at this site. The SO(4)(2-) binding structure supported the single-pathway kinetic model.     

Kinetic studies show that Ca2+ and Tb3+ have different binding preferences toward the four Ca2+-binding sites of calmodulin

The stepwise addition of Tb3+ to calmodulin yields a large tyrosine-sensitized Tb3+ luminescence enhancement as the third and fourth ions bind to the protein [Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., & Gergely, J. (1982) Eur. J. Biochem. 124, 7-12]. Since the only tyrosine residues in calmodulin are located within binding sites III and IV, these results suggest that Tb3+ binds first to sites I and II. Recent NMR studies have provided evidence that Ca2+, on the other hand, binds preferentially to sites III and IV. Kinetic studies using a stopped-flow apparatus also show that the preferential binding of Ca2+ and lanthanide ions is different. Upon rapid mixing of 2Ca-calmodulin with two Tb3+ ions, there was a small and rapid tyrosine fluorescence change, but no Tb3+ luminescence was observed, indicating that Tb3+ binds to sites I and II but not sites III and IV. When two Tb3+ ions are mixed with 2Dy-calmodulin, Tb3+ luminescence rises rapidly as Tb3+ binds to the empty sites III and IV, followed by a more gradual decrease (k = 0.4 s-1 as the ions redistribute themselves over the four sites. These results indicate that (i) both Tb3+ and Dy3+ prefer binding to sites I and II of calmodulin and (ii) the binding of Tb3+ to calmodulin is not impeded by the presence of two Ca2+ ions initially bound to the protein. Thus, the Ca2+ and lanthanide ions must exhibit opposite preferences for the four sites of calmodulin: sites III and IV are the high-affinity sites for Ca2+, whereas Tb3+ and Dy3+ prefer sites I and II.     

The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants

The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe(3+) transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe(3+) through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled (55)Fe(3+) transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.     

Fluorescence studies on the nucleotide- and Ca2+-binding domains of molluscan myosin.

The effects of nucleotides and Ca2+ on the intrinsic tryptophan fluorescence of molluscan myosin and its proteolytic fragments were studied. By using these proteins from the scallop, Pecten maximus, the existence of two distinct tryptophan-containing domains was established, which respond independently to ATP and Ca2+-specific binding. The latter is located in the 'neck' region of the myosin, which constitutes the regulatory domain. Subfragment 1, lacking the regulatory domain, responded only to ATP binding. On the other hand a tryptic fragment comprising the regulatory domain responded only to Ca2+ binding. Subfragment 1, containing the regulatory domain, responded to both ATP and Ca2+, but its ATPase activity was Ca2+-insensitive. By contrast, the ATPase activity of HMM was Ca2+-sensitive. Increasing the ionic strength had a detrimental effect on Ca2+-sensitivity, and fluorescence studies on solubilized myosin were therefore of limited value. Myosin and its fragments from other molluscan species which were investigated produced similar changes to those of Pectan maximus.     

Critical and distinct roles of amino- and carboxyl-terminal sequences in regulation of the biological activity of the Chp atypical Rho GTPase

Chp (Cdc42 homologous protein) shares significant sequence and functional identity with the human Cdc42 small GTPase, and like Cdc42, promotes formation of filopodia and activates the p21-activated kinase serine/threonine kinase. However, unlike Cdc42, Chp contains unique amino- and carboxyl-terminal extensions. Here we determined whether Chp, like Cdc42, can promote growth transformation and evaluated the role of the amino- and carboxyl-terminal sequences in Chp function. Surprisingly, we found that a GTPase-deficient mutant of Chp exhibited low transforming activity but that deletion of the amino terminus of Chp greatly enhanced its transforming activity. Thus, the amino terminus may serve as a negative regulator of Chp function. The carboxyl terminus of Cdc42 contains a CAAX (where C is cysteine, A is aliphatic amino acid, X is terminal amino acid) tetrapeptide sequence that signals for the posttranslational modification critical for Cdc42 membrane association and biological function. Although Chp lacks aCAAXmotif, we found that Chp showed carboxyl terminus-dependent localization to the plasma membrane and to endosomes. Furthermore, an intact carboxyl terminus was required for Chp transforming activity. However, treatment with inhibitors of protein palmitoylation, but not prenylation, caused Chp to mislocalize to the cytoplasm. Thus, Chp depends on palmitoylation, rather than isoprenylation, for membrane association and function. In summary, Chp is implicated in cell transformation, and the unique amino and carboxyl termini of Chp represent atypical mechanisms of regulation of Rho GTPase function.     

高岭土对Fe~(2+)的吸附及其对高岭土中Fe~(3+)生物还原的影响研究

利用异化铁还原微生物可将高岭土中不溶性的Fe3+还原成可溶性的Fe2+,但是此过程中产生的Fe2+能够被高岭土以及异化铁还原微生物吸附,从而影响高岭土中铁的异化还原。本文研究了pH、高岭土量、Fe2+浓度、温度4个因素对高岭土吸附Fe2+的影响;并采用Logistic方程拟合,研究Fe2+及温度对高岭土中Fe3+的生物还原特征。结果表明:pH、高岭土量、浓度、温度4个因素均会影响高岭土吸附Fe2+,当Fe2+吸附在高岭土和微生物菌体表面时,微生物的活性下降,同时高岭土表面Fe3+的生物可利用性也降低,Fe3+生物还原的最大速率减小。     

Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin.

Skeletal-muscle UDP-glucose pyrophosphorylase is inactivated by reaction with 2-ethoxy-N-(ethoxy-carbonyl)-1,2-dihydroquinoline (EEDQ) and 1-(3-dimethylaminopropyl-3-ethylcarbodi-imide (EDAC), two reagents specific for carboxylate groups. The former reagent is a more effective inactivator than EDAC. Although no evidence of reversible enzyme-reagent complexes of the affinity-labelling type was obtained by kinetic analysis of the inactivation, the selective protection of UDP-glucose pyrophosphorylase activity against inactivation by EEDQ in the presence of uridine substrates is indicative of an active-site-directed effect. The results are consistent with the hypothesis that EEDQ modifies a single carboxylate group located in a hydrophobic domain close to the substrate-binding site, leading to enzyme inactivation. In contrast, the reaction between UDP-glucose pyrophosphorylase and EDAC appears to involve a different region of the enzyme.     

Distinct roles for amino- and carboxyl-terminal sequences of SPRR1 protein in the formation of cross-linked envelopes of conducting airway epithelial cells

The small proline-rich protein, SPRR1, is a marker gene whose expression in conducting airway epithelium is elevated under a variety of conditions that enhance squamous differentiation. The purpose of this study is to elucidate the nature of the SPRR1 sequence involved in cross-linked envelope formation in a tissue/cell type, such as conducting airway epithelium, that normally does not express squamous function except after injury or maintenance in culture. For this, a Flag-SPRR1 fusion protein expression system has been developed. Using the liposome-mediated gene transfer technique on passage 1 culture of human tracheobronchial epithelial (TBE) cells, the Flag-SPRR1 fusion protein can be expressed and detected immunologically by both anti-Flag and anti-SPRR1 antibodies. The incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes can be demonstrated when transfected human passage 1 TBE cultures are treated with phorbol 12-myristate 13-acetate and high calcium (1.5 mM). By deletion and site-directed mutagenesis, two distinct roles of the amino- and carboxyl-terminal sequences of SPRR1 have been demonstrated. First, we demonstrated that the amino-terminal sequence of SPRR1 protein is required for the incorporation of the fusion protein into cross-linked envelopes, whereas a deletion on the carboxyl-terminal region or on the middle repetitive unit has no effect. Interestingly, insertion of a 24-amino acid peptide of monkey MUC2 repetitive sequence in the amino-terminus of SPRR1 protein had a stimulatory effect. Site-directed mutagenesis on the following amino acid residues, Lys(7), Gln(88), and Lys(89), which were found previously to participate in the cross-linked envelope formation of keratinocytes, had no detrimental effect on the incorporation. However, mutations on Gln clusters, such as Gln(4)-Gln(6) and Gln(22)-Gln(25), had detrimental effects on the incorporation. These results suggest an amino-terminal sequence-dependent and multiple cross-linked sites for the incorporation of Flag-SPRR1 fusion protein into cross-linked envelopes of cultured human TBE cells. Second, we demonstrated that the carboxyl terminus of SPRR1 protein is required for a high level of Flag-fusion protein expression. A deletion in the carboxyl region or a mutation on the last lysine residue of the carboxyl end had a detrimental effect on the level of Flag-SPRR1 fusion protein expressed in transfected cells. In contrast, there was only a slight decrease in the level of expression if the amino-terminus was deleted. Interestingly, the efficiency for fusion protein to incorporate into cross-linked envelopes was elevated by the mutation at the carboxyl end. These results suggest distinct roles, perhaps coordinately, for both amino- and carboxyl-terminal sequences in the regulation of the life cycle of SPRR1 protein in cultured TBE cells.     

Rhizosphere acidification and Fe3+ reduction in lupins and peas: Iron deficiency in lupins is not due to a poor ability to reduce Fe3+

While lupins suffer severely from Fe deficiency when grown on calcareous soils, field peas under the same conditions grow normally. This paper aimed to identify whether these differences were related to differences in either the pattern or capacity for rhizosphere acidification or Fe³⁺ reduction between these species. Two lupin species (Lupinus angustifolius, L. cosentinii) and field peas (Pisum sativum) were grown in solution culture for 5 weeks with both an adequate and a low supply of Fe. Plants were reliant on symbiotically fixed N. The extent of iron reduction was determined using the chelates TPTZ and BPDS. The pattern of reactions around roots was determined by placing roots in agar containing either bromocresol purple or TPTZ. The low supply of Fe decreased the growth of lupins by over 30% and induced severe chlorosis and necrosis. Growth of the peas was reduced by less than 15% and no symptoms appeared. All species acidified the solutions by about 1 pH unit regardless of the Fe treatment. The level of Fe³⁺ reduction was higher for all species grown with low Fe than with adequate Fe. Capacity for Fe³⁺ reduction was higher for all species grown with low Fe than with adequate Fe. Capacity for Fe³⁺ reduction was similar for all species. The pattern of acidification and reduction around roots was also similar between species. Thus it appears that the capacity of lupins to reduce Fe³⁺ in the rhizosphere is not the primary cause of Fe deficiency in lupins.     

Mössbauer spectroscopy and electron paramagnetic resonance studies of iron metabolites inPseudomonas aeruginosa: Fe2+ and Fe3+ ferritin in57ferripyoverdine incubated cells and57ferric citrate fed cells

Pseudomonas aeruginosa samples were studied using Mössbauer spectroscopy and electron paramagnetic resonance (EPR). Samples included whole cells, membranes, and soluble fractions from cells which had been grown with⁵⁷ferric chloride,⁵⁷ferric citrate or incubated with⁵⁷ferripyoverdine. These experiments show for the first time thatP. aeruginosa can accumulate iron in a bacterioferritin when grown under conditions of iron limitation and incubated with its cognate ferrisiderophore, ferripyoverdine. Soluble fraction fromP. aeruginosa cells which were grown iron starved and incubated with⁵⁷ferripyoverdine for 120 min showed the presence of both a ferric and ferrous complex whose Mössbauer spectra matched that of bacterioferritin extracted fromAzotobacter vinelandii and whose EPR spectra showed a characteristic ferritin-like resonance. A second soluble fraction sample from cells which had been grown with⁵⁷ferric citrate also showed the presence of a species with the same EPR and Mössbauer parameters. In addition Western blotting confirmed the presence of bacterioferritin in the soluble fraction of the cells which had been incubated with ferripyoverdine.     

The amino- and carboxyl-terminal fragments of the Bacillus thuringensis Cyt1Aa toxin have differential roles in toxin oligomerization and pore formation

The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a β-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the β-sheet, allowing insertion of β-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of β-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane.     

Cell degeneration in the developing optic lobes of the sine oculis and small-optic-lobes mutants of Drosophila melanogaster

In the small-optic-lobes (sol) and sine oculis (so) mutants of Drosophila melanogaster extensive cell death occurs in the optic lobes during the first half of pupal development. Gynandromorph flies show that the sol mutation acts primarily on cells of the medulla cortex. Degeneration of medullar ganglion cells occurs at an early stage of cellular differentiation, when their axons have not yet participated in the formation of the second optic chiasma. The so gene, on the other hand, acts on the eye anlagen. The analysis of chimeric flies demonstrates that degeneration in the optic lobes of so flies is a consequence of eye reduction. At the level of the second optic chiasma extensive axonal degeneration can be observed in the mutant. Neurons seem to die after their failure to establish a sufficient number of functional contacts. In sol;so double mutants, the mutational effects are cumulative causing complete degeneration of columnar cell types in pupae without any eye anlage. The tiny rudiments of the optic lobes in eyeless double mutants still contain tangential neurons of the medulla and of the lobula complex. The central brain is reduced in size due to the missing visual fibers, however, its overall appearance is surprisingly normal.