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J M Mauro

Publications and source records attributed to J M Mauro.

At least 19 recordsLinked to original sources
PubMed2004

A fluorescence resonance energy transfer-derived structure of a quantum dot-protein bioconjugate nanoassembly.

The first generation of luminescent semiconductor quantum dot (QD)-based hybrid inorganic biomaterials and sensors is now being developed. It is crucial to understand how bioreceptors, especially proteins, interact with these inorganic nanomaterials. As a model system for study, we use Rhodamine red-labeled engineered variants of Escherichia coli maltose-binding protein (MBP) coordinated to the surface of 555-nm emitting CdSe-ZnS core-shell QDs. Fluorescence resonance energy transfer studies were performed to determine the distance from each of six unique MBP-Rhodamine red dye-acceptor locations to the center of the energy-donating QD. In a strategy analogous to a nanoscale global positioning system determination, we use the intraassembly distances determined from the fluorescence resonance energy transfer measurements, the MBP crystallographic coordinates, and a least-squares approach to determine the orientation of the MBP relative to the QD surface. Results indicate that MBP has a preferred orientation on the QD surface. The refined model is in agreement with other evidence, which indicates coordination of the protein to the QD occurs by means of its C-terminal pentahistidine tail, and the size of the QD estimated from the model is in good agreement with physical measurements of QD size. The approach detailed here may be useful in determining the orientation of proteins in other hybrid protein-nanoparticle materials. To our knowledge, this is the first structural model of a hybrid luminescent QD-protein receptor assembly elucidated by using spectroscopic measurements in conjunction with crystallographic and other data.

Carrier Proteins↗
PubMed2000

Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.

Amino Acid Sequence↗
PubMed2000

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR.

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H2O vs. D2O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.

Alanine↗
PubMed2000

Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene.

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and targeted to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind 109Cd2+. The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.

ATP-Binding Cassette Transporters↗
PubMed1998

Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine.

The current human whole-cell vaccine is ineffective against pneumonic plague caused by typical F1 capsule positive (F1+) strains of Yersinia pestis. The authors found this vaccine to also be ineffective against F1-negative (F1-) Y. pestis strains, which have been isolated from a human case and from rodents. For these reasons, the authors developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. This vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either an F1+ or F1- strain of Y. pestis, gave better protection than F1 or V alone against the F1+ strain, and may provide the basis for an improved human plague vaccine.

Aerosols↗
PubMed1996

Fiber-optic fluorometric sensing of polymerase chain reaction-amplified DNA using an immobilized DNA capture protein.

A fiber-optic assay for amplified DNA products has been developed. Modifications of the DNA capture strategy described previously by Kemp et al. [Proc. Natl. Acad. Sci. USA 86, 2423-2427 (1989)] were made that allowed selective binding of DNA labeled during the amplification process to the sensing surface of fused silica fibers. The gene for a chimeric protein composed of the IgG-binding beta 2 subdomain of streptococcal protein G fused with the DNA binding domain of yeast GCN4 was constructed, and this PG/GCN4 protein was overexpressed in Escherichia coli. The purified protein was noncovalently bound to IgG-modified fibers utilizing strong and specific interactions between the protein G beta 2 domain and goat IgG that had been covalently immobilized on the fiber surface. Nanomolar concentrations of amplified DNA labeled with the fluorophore tetramethylrhodamine and the AP-1 consensus nucleotide sequence recognized by GCN4 (5'-ATGACTCAT) were rapidly and selectively bound within the evanescent zone of multimode laser-illuminated fibers. Signal from unincorporated fluorescent PCR primer was negligible. Individual fibers could be used for multiple sequential assays, since the fluorescent double-stranded DNA was rapidly and completely stripped from their surfaces with high salt solutions, leaving the IgG-PG/GCN4 DNA binding complex intact to accept another PCR sample.

Amino Acid Sequence↗
PubMed1995

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants.

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.

Binding Sites↗
PubMed1995

Preliminary crystallographic analysis of chloroperoxidase from Caldariomyces fumago.

Chloroperoxidase from the fungus Caldariomyces fumago has been crystallized in two space groups, C222(1) and P2(1)2(1)2(1) both of which are suitable for high-resolution X-ray studies. Parent data sets have been obtained to 2.16 A in space group C222(1) and 2.00 A in space group P2(1)2(1)2(1). Heavy-atom derivatives have been obtained with both forms and electron-density maps calculated. The heme has been located and continuous electron density between the heme and protein clearly indicates the location of the proximal ligand.

Journal Article↗
PubMed1995

Expression of the Neurospora crassa metallothionein gene in Escherichia coli and its effect on heavy-metal uptake.

The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.

ATP-Binding Cassette Transporters↗
PubMed1994

Thermodynamics of hydrogen cyanide and hydrogen fluoride binding to cytochrome c peroxidase and its Asn-82-->Asp mutant.

The thermodynamics of binding of fluoride and cyanide to cytochrome c peroxidase (CCP) and its Asn-82-->Asp mutant (D82CCP) in phosphate and acetate buffer at an ionic strength of 0.15 mol.kg-1 from pH 5.0 to 7.1 were investigated by titration calorimetry at 289 and 297 K. The binding reactions are enthalpically driven. The fluoride-binding constants determined from the titration calorimetry results were in agreement with those determined from difference-spectroscopy measurements. For cyanide binding to CCP at 297.9 K, the binding constant decreased from 8.95 (+/- 0.83) x 10(5) M-1 at pH 7.0 to 4.04(+/- 0.23) x 10(5) M-1 at pH 5.0, and the binding enthalpy increased from -57.2 +/- 1.4 kJ.mol-1 at pH 7.0 to -48.6 +/- 1.8 kJ.mol-1 at pH 5.0. For fluoride binding to CCP, the binding constant increased from 8.41(+/- 0.54) x 10(3) M-1 at pH 7.0 to 3.11(+/- 0.09) x 10(5) M-1 at pH 5.0 and the binding enthalpy increased from -71.9 +/- 1.1 kJ.mol-1 at pH 7.0 to -67.0 +/- 1.9 kJ.mol-1 at pH 5.0. The binding enthalpies for D82CCP were about the same as those for CCP. However, the binding constants for cyanide and fluoride to D82CCP were respectively a factor of two less and at least an order of magnitude less than the corresponding binding constants of CCP. Decreased ligand-binding strength in the D82CCP mutant is thus entirely due to entropic effects.

Asparagine↗
PubMed1994

The effect of the Asn82-->Asp mutation in yeast cytochrome c peroxidase studied by proton NMR spectroscopy.

Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase-cyanide with the Asn 82-->Asp mutation ([N82D]cytochrome c peroxidase-CN) are presented and compared to the wild-type enzyme. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase. In wild-type cytochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The principal result from this study is the observation that three hyperfine-shifted resonances disappear from the spectrum of [N82D] cytochrome c peroxidase-CN compared to the wild-type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase-CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52-Asn82 and His52-heme-coordinated cyanide hydrogen bonds.

Asparagine↗
PubMed1992

Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.

Amino Acid Sequence↗
PubMed1992

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.

Asparagine↗
PubMed1992

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗
PubMed1991

Compound I radical in site-directed mutants of cytochrome c peroxidase as probed by electron paramagnetic resonance and electron-nuclear double resonance.

The reaction of ferric cytochrome c peroxidase (CcP) from Saccharomyces cerevisiae with peroxide produces compound I, characterized by both an oxyferryl iron center and a protein-based free radical. The electron paramagnetic resonance (EPR) signal of the CcP compound I radical can be resolved into a broad majority component which accounts for approximately 90% of the spin intensity and a narrow minority component which accounts for approximately 10% of the integrated spin intensity [Hori, H., & Yonetani, T. (1985) J. Biol. Chem. 260, 3549-3555]. It was shown previously that the broad component of the compound I radical signal is eliminated by mutation of Trp-191 to Phe [Scholes, C. P., Liu, Y., Fishel, L. F., Farnum, M. F., Mauro, J. M., & Kraut, J. (1989) Isr. J. Chem. 29, 85-92]. The present work probed the effect of mutations in the vicinity of this residue by EPR and electron-nuclear double resonance (ENDOR). These mutations were obtained from a plasmid-encoded form of S. cerevisiae expressed in Escherichia coli [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360]. The EPR line shape and ENDOR signals of the compound I radical were perturbed only by mutations that alter Trp-191 or residues in its immediate vicinity: namely, Met-230 and Met-231, which have sulfur atoms within 4 A of the indole ring, and Asp-235, which forms a hydrogen bond with the indole nitrogen of Trp-191. Mutations of other potential oxidizable sites (tryptophan, tyrosine, methionine, and cysteine) did not alter the EPR line shapes of the compound I radical, although the integrated spin intensities were weaker in some of these mutants. Mutations at Met-230 and/or -231 perturbed the EPR line shapes of the compound I radical signal but did not eliminate it. ENDOR of these two methionine mutants showed alteration to the hyperfine couplings of several strongly coupled protons, which are characteristic of the majority compound I radical electronic structure, and a change in weaker hyperfine couplings, which suggests a different orientation of the radical with respect to its surroundings in the presence of these methionine mutations. Besides the Trp-191----Phe mutation, only the Asp-235----Asn mutation eliminated the broad component of the compound I signal. Loss of the broad compound I EPR signal coincides with both the loss of the Asp----Trp-191 hydrogen-bonding interaction and alteration of the position of the indole ring of Trp-191.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

CO dissociation in cytochrome c peroxidase: site-directed mutagenesis shows that distal Arg 48 influences CO dissociation rates.

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine↗
PubMed1990

Comparative proton NMR analysis of wild-type cytochrome c peroxidase from yeast, the recombinant enzyme from Escherichia coli, and an Asp-235----Asn-235 mutant.

Proton NMR spectra of cytochrome c peroxidase (CcP) isolated from yeast (wild type) and two Escherichia coli expressed proteins, the parent expressed protein [CcP(MI)] and the site-directed mutant CcP(MI,D235N) (Asp-235----Asn-235), have been examined. At neutral pH and in the presence of only potassium phosphate buffer and potassium nitrate, wild-type Ccp and CcP(MI) demonstrate nearly identical spectra corresponding to normal (i.e., "unaged") high-spin ferric peroxidase. In contrast, the mutant protein displays a spectrum characteristic of a low-spin form, probably a result of hydroxide ligation. Asp-235 is hydrogen-bonded to the proximal heme ligand, His-175. Changing Asp-235 to Asn results in alteration of the pK for formation of the basic form of CcP. Thus, changes in proximal side structure mediate the chemistry of the distal ligand binding site. All three proteins bind F-, N3-, and CN- ions, although the affinity of the mutant protein (D235N) for fluoride ion appears to be much higher than that of the other two proteins. Analysis of proton NMR spectra of the cyanide ligated forms leads to the conclusion that the mutant protein (D235N) possesses a more neutral proximal histidine imidazole ring than does either wild-type CcP or CcP(MI). It confirms that an important feature of the cytochrome c peroxidase structure is at least partial, and probably full, imidazolate character for the proximal histidine (His-175).

Asparagine↗