Structural evidence for guanidine-protein side chain interactions: crystal structure of CutA from Pyrococcus horikoshii in 3 M guanidine hydrochloride

This study was carried out to investigate the structural perturbation of the protein's local structure by the denaturants under non-denaturing conditions. Crystal structure of CutA from an archaeon Pyrococcus horikosii (PhoCutA), a heavy-metal binding protein, was determined at 1.6-angstroms resolution in the presence of 3 M guanidine HCl (GdnHCl). Native PhoCutA has a large number of short intramolecular hydrogen bonds and salt bridges on the protein surface, of which greater than 90% of hydrogen bonds and all salt bridges were retained in 3 M GdnHCl. Hydrogen bonds that disappeared in the GdnHCl crystal structure were mainly located on the protein surface, especially around the structurally perturbed loop, suggesting interactions between peptide groups and GdnHCl. Only a few GdnH+ ions were observed in the crystal structure, although none at the surface, of the protein. Two GdnH+ ions were observed in the center of the trimeric structure, replacing water molecules, and were hydrogen bonded with Asp84 and Asp86 of each chain. The exterior loop from Tyr39 to Lys44, including Trp40-Trp41, was perturbed structurally. Decreases in temperature factors were observed in beta strand 5 and the N terminus of helix 3. These results suggest the specific bindings of GdnH+ with some acidic residues and the non-specific bindings around Trp residues and peptide groups on the protein surface and that binding of GdnHCl to the native protein is limited, resulting in local structural perturbation.     

Single particle analysis of manganese-induced prion protein aggregates

Prion diseases are characterized by the conversion of the cellular prion protein (PrP(C)) to a disease-specific aggregated isoform (PrP(Sc)). We have shown that Mn(2+) ions amplify aggregation, whereas Cu(2+) has an inhibitory effect. To characterize Mn(2+)-induced aggregates, we used cross-correlation analysis as well as scanning for intensely fluorescent targets in an SDS-dependent aggregation assay with fluorescently labeled PrP. We found that the effect of Mn(2+) was mainly due to the association of preformed PrP oligomers to larger aggregates, rapidly reversible by EDTA, and independent of the histidine-dependent copper-binding sites of PrP, suggesting that Mn(2+) induces reversible intermolecular binding. In contrast, the inhibitory effect of Cu(2+) required binding to histidine-containing binding sites, indicating that binding of copper affects the structure of PrP(C) which in turn modifies the susceptibility to manganese and the ability to aggregate. These findings suggest that copper and manganese may also affect prion propagation in vivo.     

Discovery of native metal ion sites located on the ferredoxin docking side of photosystem I

Photosystem I (PSI) is a large membrane protein that catalyzes light-driven electron transfer across the thylakoid membrane from plastocyanin located in the lumen to ferredoxin in the stroma. Metal analysis reveals that PSI isolated from the cyanobacterial membranes of Synechococcus leopoliensishas a near-stoichiometric 1 molar equiv of Zn (2+) per PSI monomer and two additional surface metal ion sites that favor Cu (2+) binding. Two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy reveals coupling to the so-called remote nitrogen of a single histidine coordinated to one of the Cu (2+) centers. EPR and X-ray absorption fine structure (XAFS) studies of 2Cu-PSI complexes reveal the direct interaction of ferredoxin with the Cu (2+) centers on PSI, establishing the location of native metal sites on the ferredoxin docking side of PSI. On the basis of these spectroscopic results and previously reported site-directed mutagenesis studies, inspection of the PSI crystal structure reveals a cluster of three highly conserved residues, His(D95), Glu(D103), and Asp(C23), as a likely Cu (2+) binding site. The discovery of surface metal sites on the acceptor side of PSI provides a unique opportunity to probe the stromal region of PSI and the interactions of PSI with its reaction partner, the soluble electron carrier protein ferredoxin.     

Effect of chemical modification of histidines on the copper-induced oligomerization of jack bean urease (EC 3.5.1.5)

Aggregation of jack bean urease (JBU) is associated with alterations of its biological properties, notably the ureolytic and entomotoxic activities. We investigated the influence of metals on protein oligomerization and biological properties. Besides protein aggregation, Cu(2+) induces inhibition of both ureolytic and insecticidal activities of JBU. Chemical modification of histidine residues in JBU with diethylpyrocarbonate (DEPC) decreases its affinity for Cu(2+) and inhibits oligomerization induced by this metal. Furthermore, this modification protects the insecticidal properties of JBU from being inactivated by Cu(2+). Although DEPC-treated JBU displayed lower ureolytic activity, the modified protein is less susceptible to inhibition by Cu(2+) when compared to native enzyme. Our findings show that Cu(2+) promotes JBU aggregation and differently of other heavy metals studied here, it apparently inhibits the ureolytic activity by inducing protein polymerization along with blockage of sulfhydryl groups.     

Ligand binding promotes prion protein aggregation--role of the octapeptide repeats

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu(2+) and Zn(2+). Here, we report our findings that GAG and Cu(2+) promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu(2+), mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP. When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu(2+) is abolished. By contrast, when the Cu(2+)-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu(2+) is able to promote aggregation. Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu(2+)-promoted aggregation. However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu(2+). Therefore, although binding of either GAG or Cu(2+) promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation.     

Effect of metal ions on de novo aggregation of full-length prion protein

It is well established that the prion protein (PrP) contains metal ion binding sites with specificity for copper. Changes in copper levels have been suggested to influence incubation time in experimental prion disease. Therefore, we studied the effect of heavy metal ions (Cu(2+), Mn(2+), Ni(2+), Co(2+), and Zn(2+)) in vitro in a model system that utilizes changes in the concentration of SDS to induce structural conversion and aggregation of recombinant PrP. To quantify and characterize PrP aggregates, we used fluorescently labelled PrP and cross-correlation analysis as well as scanning for intensely fluorescent targets in a confocal single molecule detection system. We found a specific strong pro-aggregatory effect of Mn(2+) at low micromolar concentrations that could be blocked by nanomolar concentration of Cu(2+). These findings suggest that metal ions such as copper and manganese may also affect PrP conversion in vivo.     

Copper alters aggregation behavior of prion protein and induces novel interactions between its N- and C-terminal regions

Copper is reported to promote and prevent aggregation of prion protein. Conformational and functional consequences of Cu(2+)-binding to prion protein (PrP) are not well understood largely because most of the Cu(2+)-binding studies have been performed on fragments and truncated variants of the prion protein. In this context, we set out to investigate the conformational consequences of Cu(2+)-binding to full-length prion protein (PrP) by isothermal calorimetry, NMR, and small angle x-ray scattering. In this study, we report altered aggregation behavior of full-length PrP upon binding to Cu(2+). At physiological temperature, Cu(2+) did not promote aggregation suggesting that Cu(2+) may not play a role in the aggregation of PrP at physiological temperature (37 °C). However, Cu(2+)-bound PrP aggregated at lower temperatures. This temperature-dependent process is reversible. Our results show two novel intra-protein interactions upon Cu(2+)-binding. The N-terminal region (residues 90-120 that contain the site His-96/His-111) becomes proximal to helix-1 (residues 144-147) and its nearby loop region (residues 139-143), which may be important in preventing amyloid fibril formation in the presence of Cu(2+). In addition, we observed another novel interaction between the N-terminal region comprising the octapeptide repeats (residues 60-91) and helix-2 (residues 174-185) of PrP. Small angle x-ray scattering studies of full-length PrP show significant compactness upon Cu(2+)-binding. Our results demonstrate novel long range inter-domain interactions of the N- and C-terminal regions of PrP upon Cu(2+)-binding, which might have physiological significance.     

Structural characterization of the major extrapallial fluid protein of the mollusc Mytilus edulis: implications for function

The major protein component of the extrapallial fluid of the mollusc Mytilus edulis has been previously isolated and partially characterized. It was postulated to play a role in shell mineralization because of its intriguing property of Ca(2+)-binding-induced self-assembling. However, it also binds other divalent ions, including Cd(2+), Cu(2+), Mn(2+), and Mg(2+). Herein is the initial report on the characterization of the primary structure of the extrapallial (EP) protein by RT-PCR and cDNA sequencing methods and by de novo peptide sequencing with mass spectrometry. The EP protein is comprised of 213 amino acids postcleavage of a signal peptide of 23 amino acids. The protein is rich in His, Glu, and Asp residues. The site of N-glycosylation, "NHTE", at amino acid positions 54-57 and the intramolecular disulfide bond between Cys 139 and Cys 171 of the protein have been characterized also. Sequence comparisons reveal that the EP protein possesses little homology to any presently known matrix proteins previously isolated from mollusc shells but rather it highly resembles a heavy metal binding protein and a histidine-rich glycoprotein, both from the hemolymph of M. edulis. The predicted domain profile and amino acid composition suggest that its N-terminus may be involved in calcium binding. The abundance of histidine residues of the protein may account for its heavy metal binding properties. Thus, the EP protein perhaps has multiple functions, serving as a Ca(2+)-transport protein, a shell matrix protein, and a heavy metal detoxification protein.     

Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography

Escherichia coli RNase HI has two Mn(2+)-binding sites. Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134. Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively. However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity. In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined. In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala. In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala. In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn. All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions. These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket. These positions are different from one another, but none of them is similar to site 1. The temperature factors of these manganese ions were considerably larger than those of the surrounding residues. These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets. We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model). The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion.     

Cu2+ triggers reversible aggregation of a disordered His-rich dehydrin MpDhn12 from Musa paradisiaca

Copper is an essential nutrient, but it is toxic in excess. Here, we cloned and characterized a His-rich low molecular weight dehydrin from Musa paradisiaca, MpDhn12. Analysis by circular dichroism (CD) spectra and a thermal stability assay showed that MpDhn12 is an intrinsically disordered protein, and immobilized-metal affinity chromatography (IMAC) analysis revealed that MpDhn12 can bind Cu(2+) both in vitro and in vivo. Interestingly, MpDhn12 aggregated under excess Cu(2+) conditions, and the aggregation was reversible and impaired by histidine modification with diethylpyrocarbonate (DEPC), while the disordered structure of another dehydrin ERD14 (as a control) was not changed. Furthermore, MpDhn12 could complement the copper-sensitive phenotype of yeast mutant Δsod1. These results together suggested that MpDhn12 may take part in buffering copper levels through chelation and formation of aggregates in excess Cu(2+) conditions. To the best of our knowledge, it is the first report that a dehydrin interchanged between disordered and aggregated state triggered by copper.     

Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn(2+). Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn(2+)-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of >100 microM MnCl(2), and their activities were not inhibited at higher Mn(2+) concentrations (up to 10 mM). The wild-type protein contains two Mn(2+) binding sites and is activated upon binding of one Mn(2+) ion at site 1 at low ( approximately 1 microM) Mn(2+) concentrations. This activity is attenuated upon binding of a second Mn(2+) ion at site 2 at high (>10 microM) Mn(2+) concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn(2+) concentrations, were identical to that of the wild-type protein at low Mn(2+) concentrations but were different from that of the wild-type protein at high Mn(2+) concentrations. These results suggest that one Mn(2+) ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn(2+) ion to these proteins in the absence of the substrate support this hypothesis. When Mn(2+) ion is used as a metal cofactor, the Mn(2+) ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.     

Copper and zinc binding modulates the aggregation and neurotoxic properties of the prion peptide PrP106-126.

The abnormal form of the prion protein (PrP) is believed to be responsible for the transmissible spongiform encephalopathies. A peptide encompassing residues 106-126 of human PrP (PrP106-126) is neurotoxic in vitro due its adoption of an amyloidogenic fibril structure. The Alzheimer's disease amyloid beta peptide (Abeta) also undergoes fibrillogenesis to become neurotoxic. Abeta aggregation and toxicity is highly sensitive to copper, zinc, or iron ions. We show that PrP106-126 aggregation, as assessed by turbidometry, is abolished in Chelex-100-treated buffer. ICP-MS analysis showed that the Chelex-100 treatment had reduced Cu(2+) and Zn(2+) levels approximately 3-fold. Restoring Cu(2+) and Zn(2+) to their original levels restored aggregation. Circular dichroism showed that the Chelex-100 treatment reduced the aggregated beta-sheet content of the peptide. Electron paramagnetic resonance spectroscopy identified a 2N1S1O coordination to the Cu(2+) atom, suggesting histidine 111 and methionine 109 or 112 are involved. Nuclear magnetic resonance confirmed Cu(2+) and Zn(2+) binding to His-111 and weaker binding to Met-112. An N-terminally acetylated PrP106-126 peptide did not bind Cu(2+), implicating the free amino group in metal binding. Mutagenesis of either His-111, Met-109, or Met-112 abolished PrP106-126 neurotoxicity and its ability to form fibrils. Therefore, Cu(2+) and/or Zn(2+) binding is critical for PrP106-126 aggregation and neurotoxicity.     

Paramagnetic NMR study of Cu(2+)-IDA complex localization on a protein surface and its application to elucidate long distance information

The paramagnetic metal chelate complex Cu(2+)-iminodiacetic acid (Cu(2+)-IDA) was mixed with ubiquitin, a small globular protein. Quantitative analyses of (1)H and (15)N chemical shift changes and line broadenings induced by the paramagnetic effects indicated that Cu(2+)-IDA was localized to a histidine residue (His68) on the ubiquitin surface. The distances between the backbone amide proton and the Cu(2+) relaxation center were evaluated from the proton transverse relaxation rates enhanced by the paramagnetic effect. These correlated well with the distances calculated from the crystal structure up to 20 A. Here, we show that a Cu(2+)-IDA is the first paramagnetic reagent that specifically localizes to a histidine residue on the protein surface and gives the long-range distance information.     

Structure and mechanism of 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase. An enzyme in the mevalonate-independent isoprenoid biosynthetic pathway.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.     

Destabilization of non-pathological variants of ataxin-3 by metal ions results in aggregation/fibrillogenesis

Ataxin-3 (AT3), a protein that causes spinocerebellar ataxia type 3, has a C-terminus containing a polyglutamine stretch, the length of which can be expanded in its pathological variants. Here, we report on the role of Cu(2+), Mn(2+), Zn(2+) and Al(3+) in the induction of defective protein structures and subsequent aggregation/fibrillogenesis of three different non-pathological forms of AT3, i.e. murine (Q6), human non-expanded (Q26) and human moderately expanded (Q36). AT3 variants showed an intrinsic propensity to misfolding/aggregation; on the other hand, Zn(2+) and Al(3+) strongly stimulated the amplitude and kinetics of these conformational conversions. While both metal ions induced a time-dependent aggregation into amyloid-like fibrillar forms, only small oligomers and/or short protofibrillar species were detected for AT3s alone. The rate and extent of the metal-induced aggregation/fibrillogenesis processes increased with the size of the polyglutamine stretch. Mn(2+) and Cu(2+) had no effect on (Q6) or actually prevented (Q26 and Q36) the AT3 structural transitions. The observation that Zn(2+) and Al(3+) promote AT3 fibrillogenesis is consistent with similar results found for other amyloidogenic molecules, such as beta-amyloid and prion proteins. Plausibly, these metal ions are a major common factor/cofactor in the etiopathogenesis of neurodegenerative diseases. Studies of liposomes as membrane models showed dramatic changes in the structural properties of the lipid bilayer in the presence of AT3, which were enhanced after supplementing the protein with Zn(2+) and Al(3+). This suggests that cell membranes could be a potential primary target in the ataxin-3 pathogenesis and metals could be a biological factor capable of modulating their interaction with AT3.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

The functional role of the binuclear metal center in D-aminoacylase: one-metal activation and second-metal attenuation

Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: alphabeta-binuclear, alpha-mononuclear, beta-mononuclear, and metal-independent subsets. The d-aminoacylase from Alcaligenes faecalis DA1 belongs to the beta-mononuclear subset due to the fact that the catalytically essential Zn(2+) is tightly bound at the beta site with coordination by Cys(96), His(220), and His(250), even though it possesses a binuclear active site with a weak alpha binding site. Additional Zn(2+), Cd(2+), and Cu(2+), but not Ni(2+), Co(2+), Mg(2+), Mn(2+), and Ca(2+), can inhibit enzyme activity. Crystal structures of these metal derivatives show that Zn(2+) and Cd(2+) bind at the alpha(1) subsite ligated by His(67), His(69), and Asp(366), while Cu(2+) at the alpha(2) subsite is chelated by His(67), His(69) and Cys(96). Unexpectedly, the crystal structure of the inactive H220A mutant displays that the endogenous Zn(2+) shifts to the alpha(3) subsite coordinated by His(67), His(69), Cys(96), and Asp(366), revealing that elimination of the beta site changes the coordination geometry of the alpha ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the d-aminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the beta site, but inhibited by the subsequent binding of the second ion at the alpha site.     

Aggregation/fibrillogenesis of recombinant human prion protein and Gerstmann-Sträussler-Scheinker disease peptides in the presence of metal ions

In this study we investigated the role of Cu(2+), Mn(2+), Zn(2+), and Al(3+) in inducing defective conformational rearrangements of the recombinant human prion protein (hPrP), which trigger aggregation and fibrillogenesis. The research was extended to the fragment of hPrP spanning residues 82-146, which was identified as a major component of the amyloid deposits in the brain of patients affected by Gerstmann-Str?ussler-Scheinker (GSS) disease. Variants of the 82-146 wild-type subunit [PrP-(82-146)(wt)] were also examined, including entirely, [PrP-(82-146)(scr)], and partially scrambled, [PrP-(82-146)(106)(-)(126scr)] and [PrP-(82-146)(127)(-)(146scr)], peptides. Al(3+) strongly stimulated the conversion of native hPrP into the altered conformation, and its potency in inducing aggregation was very high. Despite a lower rate and extent of prion protein conversion into altered isoforms, however, Zn(2+) was more efficient than Al(3+) in promoting organization of hPrP aggregates into well-structured, amyloid-like fibrillar filaments, whereas Mn(2+) delayed and Cu(2+) prevented the process. GSS peptides underwent the fibrillogenesis process much faster than the full-length protein. The intrinsic ability of PrP-(82-146)(wt) to form fibrillar aggregates was exalted in the presence of Zn(2+) and, to a lesser extent, of Al(3+), whereas Cu(2+) and Mn(2+) inhibited the conversion of the peptide into amyloid fibrils. Amino acid substitution in the neurotoxic core (sequence 106-126) of the 82-146 fragment reduced its amyloidogenic potential. In this case, the stimulatory effect of Zn(2+) was lower as compared to the wild-type peptide; on the contrary Al(3+) and Mn(2+) induced a higher propensity to fibrillation, which was ascribed to different binding modalities to GSS peptides. In all cases, alteration of the 127-146 sequence strongly inhibited the fibrillogenesis process, thus suggesting that integrity of the C-terminal region was essential both to confer amyloidogenic properties on GSS peptides and to activate the stimulatory potential of the metal ions.     

DsRed as a highly sensitive, selective, and reversible fluorescence-based biosensor for both Cu(+) and Cu(2+) ions

The wild type form of Red fluorescent protein (DsRed), an intrinsically fluorescent protein found in tropical corals, is found to be highly selective, reversible and sensitive for both Cu(+) and Cu(2+), with a nanomolar detection limit. The selectivity towards these ions is retained even in the presence of other heavy metal ions. The K(d) values for monovalent and divalent copper, based on single binding isotherms, are 450 and 540 nM, respectively. The wild type DsRed sensitivity to Cu(2+) (below 1 ppb) is seven orders of magnitude better than that of the related wild type Green Fluorescent protein (GFP), and it is even 40 times more sensitive than engineered mutants of GFP. Potential binding sites have been proposed, based on amino acid sequences for copper binding and the distance from the chromophore, with the aid of computer modeling.     

A heavy metal biotrap for wastewater remediation using poly-gamma-glutamic acid

Poly-gamma-glutamic acid (gamma-PGA) obtained from Bacillus licheniformis ATCC 9945 was evaluated as a potential biosorbent material for use in the removal of heavy metals from aqueous solution. Copper (Cu(2+)) was chosen as the model heavy metal used in these studies since it is extensively used by electroplating and other industries, has been the model for many other similar studies, and can be easily assayed through a number of convenient methods. Cu(2+)-gamma-PGA binding parameters under varying conditions of pH, temperature, ionic strength, and in the presence of other heavy metal ions were determined for the purified biopolymer using a specially designed dialysis apparatus. Applying the Langmuir adsorption isotherm model showed that gamma-PGA had a copper capacity approaching 77.9 mg/g and a binding constant of 32 mg/L (0.5 mM) at pH 4.0 and 25 degrees C. Cu(2+)-gamma-PGA adsorption was relatively temperature independent between 7 and 40 degrees C, while an increase in ionic strength led to a decrease in metal ion binding. Cd(2+) and Zn(2+) ions compete with Cu(2+) for binding sites on the gamma-PGA biopolymer. Metal uptake by gamma-PGA was further tested using a tangential flow filtration apparatus in a diafiltration mode in which metal was continually processed through a dilute solution of gamma-PGA without allowing for equilibrium to be established. The circulating polymer solution was able to complex metal as well as successfully prevent passage of unbound copper ions present in solution through the membrane. Using 500 mL of a 0.2% gamma-PGA solution, up to 97% of a 50 mg/L copper sulfate solution processed at a flow rate of 115 mL/min was retained by the polymer. For a 10 mg/L solution of Cu(2+) as copper sulfate, filtrate concentrations of Cu(2+) never rose above 0.6 mg/L while processing 2.5 L of dilute copper sulfate.