A catalytic site of protein disulfide isomerase probed with adenosine-5'-triphosphate analogs.

Anthraniloyl adenosine-5'-triphosphate (Ant-ATP) and etheno-adenosine-5'-triphosphate (epsilon-ATP) complexed to Mg(2+) ions are substrates of protein disulfide isomerase (PDI). epsilon-ATP, coordinated to Tb(3+) ions, was used as a probe of the ATPase binding site. Sensitized luminescence arising from resonance energy transfer from epsilon-adenine to Tb(3+) is quenched by PDI. The luminescence results are discussed in reference to a model in which the distance of separation between epsilon-adenine (donor) and Tb(3+) (acceptor) is increased upon binding of PDI. The interaction of a small peptide of 14 amino acid residues with the b/b' domain of the protein does not influence the ATPase activity. The phosphorescence, fluorescence and fluorescence anisotropy of bound epsilon-ATP are not perturbed by the binding of the small molecular weight peptide to PDI. It is suggested that the peptide and ATP do not share a common binding site on the b/b' domain.

Recognition of protein substrates by protein-disulfide isomerase. A sequence of the b' domain responds to substrate binding.

Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold molar excess of PDI over denatured protein (0. 1 microM) accelerates the recovery of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 microM. The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation constant of 0.2 microM. It is shown that the fluorescence probe is covalently attached to a SH residue located in the b' domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI.

Refolding of pyridoxine-5'-P oxidase assisted by GroEL.

The unfolding of brain pyridoxine-5'-P oxidase by guanidinium chloride has been investigated at equilibrium. Circular dichroism, fluorescence spectroscopy and gel exclusion chromatography were used to monitor the unfolding process. The enzyme dissociates reversibly into monomers, but the fluorescence properties of the cofactor FMN are not restored upon dilution with potassium phosphate buffer (pH 7.4). Spontaneous refolding leads to 20% recovery of the catalytic activity. Addition of GroEL to the renaturing buffer accelerates the recovery of catalytic activity that approaches a level of 80% with respect to the native enzyme. The rate of recovery of catalytic activity assisted by GroEL parallels the rate of FMN fluorescence quenching, suggesting that structural rearrangements of the catalytic domain is the last step to take place in the refolding process.

Retinol, a probe of conformational changes in protein disulfide isomerase.

Nanosecond and steady fluorescence techniques have been employed to study the interaction of retinol with protein disulfide isomerase (PDI). Retinol binds tightly to PDI; and the rotational correlation time (θ = 36 ns) corresponds to a monomeric subunit of 55 kDa. The enzyme does not undergo aggregation in the presence of low molecular weight peptides. Under denaturing conditions; presence of 0.75 M Gnd HCl, the fluorescence yield of bound retinol is enhanced, suggesting stronger interactions of exposed hydrophobic groups of the protein with retinol. Based on far UV CD and fluorescence measurements of the protein in the presence of Gnd HCl, it is proposed the existence of molten globule intermediates during the unfolding of PDI.

Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues.

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756-10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.

Binding of polylysine to GroEL. Inhibition of the refolding of mMDH.

Luminescence techniques have been used to investigate the interaction of GroEL with polylysine tagged with a fluorescent probe. The fluorescence emitted by anthraniloyl-polylysine, upon excitation at 320 nm, is enhanced by the addition of stoichiometric amounts of GroEL. The equilibrium dissociation constant of the complex (Kd=50 nM) was determined by fluorometric titrations. The rate and extent of recovery of the catalytic activity of denatured mitochondrial malate dehydrogenase, assisted by GroEL, is influenced by either polylysine or anthraniloyl-polylysine. It is suggested that interaction of the positively charged poly-amino acid with the apical domain of GroEL prevents binding of the unfolded protein substrate.

Refolding of thioredoxin reductase assisted by groEL and PDI.

Thioredoxin reductase was unfolded in 2 M guanidine hydrochloride as revealed by fluorescence and CD spectroscopy. Spontaneous refolding of denatured species resulted in low recovery of 10% catalytic activity after 4 h incubation at 25 degrees C. Addition of groEL or protein disulfide isomerase to the renaturation buffer accelerated the rate of recovery of catalytic activity to a level of 35 and 15%, respectively. Fluorescence spectroscopy has been used to investigate the interaction of groEL and protein disulfide isomerase with denatured thioredoxin reductase tagged with a fluorescent probe. The fluorescence emitted by the denatured protein was quenched upon binding to either groEL or protein disulfide isomerase. It is suggested that encapsulation of the protein substrate by the chaperone plays an important role in the process of folding by facilitating the formation of correctly folded species.

Recognition of partially-folded mitochondrial malate dehydrogenase by GroEL. Steady and time-dependent emission anisotropy measurements.

The binding of partially-folded mitochondrial malate dehydrogenase (mMDH) to GroEL was assessed by steady and nanosecond emission spectroscopy. Partially-folded intermediates of mMDH show significant residual secondary structure when examined by CD spectroscopy in the far UV. They bind the extrinsic fluorescent probe ANS and the protein-ANS complexes display a rotational correlation time of 19 ns. Similar rotational correlation time (phi = 18.6 ns) was determined for partially-folded species tagged with anthraniloyl. GroEL recognizes partially-folded species with a K(D) approximately 60 nM. The rotational correlation time of the complex, i.e., GroEL-mMDH-ANT, approaches a value of 280 ns in the absence of ATP. Reactivation of mMDH-ANT by addition of GroEL and ATP brings about a significant decrease in the observed rotational correlation time. The results indicate that partially-folded malate dehydrogenase is rigidly trapped by GroEL in the absence of ATP, whereas addition of ATP facilitates reactivation and release of folded conformations endowed with catalytic activity.

Porcine pyridoxal kinase c-DNA cloning, expression and confirmation of its primary sequence.

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.

Partially folded conformations of inositol monophosphatase endowed with catalytic activity.

The stability of porcine brain inositol monophosphatase in the presence of increasing concentrations of urea was investigated at pH 7.5. Exposure of the enzyme to 8 M urea brings about the dissociation of the dimeric species of 58 kDa into monomeric forms as revealed by gel filtration chromatography. Unfolding of the protein by 8 M urea results in a decrease of the ellipticity at 220 nm (20%) together with a perturbation of the near-UV circular dichroism spectrum. Urea-treated inositol monophosphatase binds Co2+ ions with a dissociation constant of 3.3 microM. The enzyme is catalytically competent when assayed with 4-nitrophenyl-phosphate in the presence of the activating ion Co2+ at pH 7.5 in 8 M urea. The apparent activation constant for Co2+ is 2.5 mM. It is postulated that partially folded conformations of monomeric species preserve their catalytic function because the affinity of Co2+ ions for the metal coordination center of the protein is not perturbed by exposure to 8 M urea.

Conformational changes at the nucleotide binding of GroEL induced by binding of protein substrates. Luminescence studies.

2'-Deoxy-3'-anthraniloyl adenosine-5-triphosphate (ANT-dATP) coordinated to Tb3+ was used as an environmentally sensitive probe of the nucleotide-binding site of GroEL. Tb3+.ANT-dATP recognizes the nucleotide-binding site of GroEL and inhibits ATPase activity. Sensitized luminescence, arising from resonance energy transfer from the anthraniloyl moiety to Tb3+, is substantially enhanced in the presence of GroEL. Binding of denatured mitochondrial malate dehydrogenase to the apical domain of GroEL causes a red shift in the fluorescence emitted by anthraniloyl and further enhancement in the phosphorescence emitted by Tb3+ upon excitation at 320 nm. It is suggested that binding of the protein substrate initiates domain movement, which is extended to the nucleotide-binding site. The luminescence results are discussed in reference to the structure of GroEL derived from x-ray crystallographic studies.

Activation of partially folded mitochondrial malate dehydrogenase by thioredoxin.

CD spectroscopy reveals that mitochondrial malate dehydrogenase in 3M guanidinium chloride shows little residual secondary structure. Refolding of the denatured protein by dilution with buffer of pH 7.5 does not restore the CD spectrum of the native enzyme. A partially folded intermediate, possessing 25% of the alpha-helix content of the native enzyme, is formed upon dilution. The partially folded intermediate binds the extrinsic probe 1-anilinonaphtalene-8-sulfonate, and the increase in fluorescence (tenfold) is accompanied by a blue shift in the band position of the emission spectrum. Partially folded malate dehydrogenase is devoid of catalytic activity. In vitro refolding of the denatured protein takes place in the presence of dithiotreitol and thioredoxin. In the presence of micromolar concentrations of thioredoxin, a recovery of approximately 70% of the catalytic activity was observed. Emission-anisotropy titrations of oxidized thioredoxin, tagged with a fluorescent probe, revealed that the oxidoreductase recognizes partially folded intermediates of malate dehydrogenase with a dissociation constant of 6 microM. Moreover, a covalently linked complex formed by thioredoxin and monomeric malate dehydrogenase was detected by SDS/PAGE. A general mechanism is postulated for the reactivation of denatured proteins by thioredoxin.

Recombinant brain 4-aminobutyrate aminotransferases overexpression, purification, and identification of Lys-330 at the active site.

4-Aminobutyrate aminotransferase (4-aminobutyrate: 2-oxoglutarate aminotransferase EC 2.6.1.19) is a key enzyme of the 4-aminobutyric acid shunt. It catalyzes the conversion of 4-aminobutyrate to succinic semialdehyde. In an effort to clarify the structure-function relationships of 4-aminobutyrate aminotransferase, we analyzed 4-aminobutyrate aminotransferase cDNA from pig brain. The inclusion bodies were formed when recombinant 4-aminobutyrate aminotransferase was overexpressed in Escherichia coli. The unfolded overproduced proteins, were purified by hydroxylapatite chromatography in the presence of urea and refolded by a sequential dialysis method. The renatured protein regained its catalytic activity. The lysyl residue at the 330 position of the amino-acid sequence serves as the anchoring site of the cofactor pyridoxal 5'-P. To verify the catalytic site of 4-aminobutyrate aminotransferase, lysine 330 was mutated to arginine by site-specific mutagenesis. Overexpression and purification of the mutated 4-aminobutyrate aminotransferase (K330R) were performed by the same method used the purification of wild-type 4-aminobutyrate aminotransferase. The purified and renatured K330R protein did not show the catalytic activity of wild type 4-aminobutyrate aminotransferase. Furthermore, the mutated protein did not show any absorption band over the spectral range of 320-460 nm characteristic of pyridoxal 5'-P covalently linked to the protein. From the results presented here, it is concluded that lysine 330 is essential for the catalytic function of the aminotransferase.

Binding of the activating ion Co(II) to myo-inositol monophosphatase monitored by fluorescence and phosphorescence spectroscopy.

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 microM, whereas the apparent activation constant Ka is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 A of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 microM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.

Reversible denaturation of myo-inositol monophosphatase. The stability of the metal-binding loop.

The unfolding of bovine brain myo-inositol monophosphatase by guanidine. HCl (Gdn. HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn.HCl-treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn.HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process. The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal-binding loop. Denaturation of the enzyme by Gdn.HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn.HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1 x 10(6)M-1. It is suggested that a residual structure of denatured myo-inositol monophosphatase is responsible for the binding of terbium ions. The kinetics of unfolding and refolding as a function of Gdn.HCl concentration were monitored by protein fluorescence in a stopped-flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80-65 s-1 at 25 degrees C. The refolding kinetics fit monoexponential functions with rate constants in the range 120-65 s-1 depending on the Gdn.HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.

Structural changes of beta-lactoglobulin B induced by urea. Evidence of residual structure.

Several spectroscopic methods have been used to study the structure of beta-lactoglobulin B at pH 2.1 in the presence of 8M urea. Fluorescence and polarization of fluorescence spectroscopy measurements indicate that the two tryptophanyl residues of the protein are exposed to the solvent in the denatured state. CD in the far-UV indicates that the amount of secondary structure in the denatured state is comparable to that found in the native state, whereas the CD spectrum in the near-UV shows that the tertiary structure is not completely disordered. The results of one-dimensional 1H NMR spectroscopy show that some local non-random structure is maintained in the denatured state, but most of the polypeptide chain has an extended non-globular conformation under the conditions of the present experiments. This conclusion is reinforced by the results of two-dimensional 1H NMR conducted on denatured samples of beta-lactoglobulin B. The study of states with intermediate levels of order will aid the understanding of how the native structure of beta-lactoglobulin B is organised during the refolding pathways.

Spectroscopic studies of myo-inositol monophosphatase with a novel fluorescent substrate.

myo-Inositol monophosphatase catalyzes dephosphorylation of the synthetic substrate anthraniloyl-2'-AMP. Binding of this fluorescent substrate to Tb(III)-monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10 angstroms. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.

Binding of a fluorescent nucleotide analog to Hsc70. The effect of peptide protein interactions on the luminescence properties of the probe.

2'-Deoxy-3'-anthraniloyladenosine-5-triphosphate(Ant-dATP) was used as an environmentally sensitive probe of the nucleotide-binding site of the molecular chaperone Hsc70. When coordinated to the lanthanide ion Tb3+, Ant-dATP is not hydrolyzed by Hsc70. The lanthanide ion acts as strong competitive inhibitor with respect to Mg2+ (Ki = 0.1 microM). Tb.Ant-dATP recognizes the nucleotide site of Hsc70 as revealed by an increase in the emission anisotropy from 0.03 to 0.21 and by a change in the fluorescence-decay time from 2.52 ns to 3.75 ns. Sensitized luminescence arising from resonance energy transfer from the anthraniloyl group to Tb3+ is substantially enhanced in the presence of Hsc70. Binding of a 20-amino-acid-residue peptide (Rnase-S peptide) to Hsc70 causes a blue shift in the fluorescence spectrum of Ant-dATP and enhances Tb3+ luminescence upon excitation at 330 nm. It is postulated that binding of the peptide to the COOH-terminal domain of Hsc70 initiates domain movement and the structural changes might extend to the nucleotide-binding site.

Screening and sequence determination of a cDNA encoding the human brain 4-aminobutyrate aminotransferase.

A human brain cDNA library constructed in the lambda ZAP II vector was screened using a fragment of pig brain cDNA encoding 4-aminobutyrate aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.

Characterization of monomeric 4-aminobutyrate aminotransferase at low pH.

4-Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, phi obs = 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL approximately 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-aminobutyrate aminotransferase in the cell.