Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli

Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol. In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol. Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation. To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli. The purified E. coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine. Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence. They revealed that E. coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1. Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent. The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment. These studies suggest that E. coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.     

Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC

Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.     

Construction and Characterization of an Escherichia coli Strain Genetically Engineered for Ni(II) Bioaccumulation

An Escherichia coli strain that accumulated Ni(II) was constructed by introducing the nixA gene (coding for a nickel transport system) from Helicobacter pylori into JM109 cells that expressed a glutathione S-transferase–pea metallothionein fusion protein. The resulting strain accumulated 15 μmol of Ni(II) per g (dry weight) from a 10 μM Ni(II) solution, four times the level taken up by JM109 cells. Ni(II) accumulation did not require an energy source, was inhibited by only 50% by 0.1 M NaCl, and occurred over the pH range from 3 to 9.     

Structural and biological analysis of the metal sites of Escherichia coli hydrogenase accessory protein HypB

The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.     

EPR investigation of the Mn(II) binding sites in glutamine synthetase (Escherichia coli W). II. Intermediate-affinity binding sites.

The nature of the intermediate-affinity (n2) Mn(II) binding sites in glutamine synthetase [EC 6.3.1.2] has been studied as a function of adenylylation in a variety of enzyme-metal complexes by EPR. In the absence of nucleotide the n2 Mn(II) environment is nearly isotropic, the Mn(II) bonds are highly ionic, and the interaction distance R greater than or equal to 12-14 A. Nucleotide binding at the n2 Mn(II) site renders the n2 Mn(II) signal unobservable and causes a reduction in signal amplitude (approximately 30%) and line broadening (approximately 6 G) at the high-affinity (n1) Mn(II) site. This behavior indicates that nucleotide binding induces a conformational change in the enzyme which brings the previously distant n1 and n2 sites into closer proximity (R less than or equal to 8-11 A), possibly for the purpose of activating the nucleotide for direct phosphoryl transfer to L-glutamate. In line with this suggestion, the broad, unresolved resonances in complexes containing both L-methionine SR-sulfoximine (MSOX) and nucleotide may result from the phosphorylation of MSOX. The n2 Mn(II) site is not affected by adenylylation in all the enzyme-metal complexes studied, which suggests that the regulatory effects of adenylylation may only act at the n1 Mn(II) sites.     

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli.

We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 microM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.     

Characterization of nine monoclonal antibodies against the Escherichia coli cyclic AMP receptor protein

Nine hybridoma clones producing antibodies against the Escherichia coli cAMP receptor protein (CRP) have been isolated. Five of the monoclonal antibodies (Class I) had a much higher affinity for native CRP while the remaining four (Class II) bound equally well to native or denatured CRP. Using native N-terminal CRP cores, it was shown that none of the Class I monoclonal antibodies cross-reacted with the 15,000-Da CRP core, and only two bound to the 18,800-Da CRP core. The positions of the antigenic determinants for the Class II monoclonal antibodies were found by Western blotting analysis to reside in the N-proximal region of CRP. Only one monoclonal antibody strongly inhibited cAMP binding by CRP, and this was accompanied by a consequent strong inhibition of both lac DNA binding and abortive initiation by RNA polymerase. Each of the Class I monoclonal antibodies inhibited abortive initiation, and four of these antibodies also blocked the binding of cAMP X CRP to the lac DNA fragment. One Class I and one Class II monoclonal antibody bound to the cAMP X CRP X DNA complex. Two of the Class II monoclonal antibodies were without apparent effect on any of the assays used.     

Subunit localizations of zinc(II) in DNA-dependent RNA polymerase from Escherichia coli B.

RNA Polymerase holoenzyme and core enzyme from Escherichia coli B have been shown to contain two zinc ions. Flameless atomic absorption spectroscopy of the isolated core subunits indicated that one zinc ion is localized on the beta subunit and the other is bound on the beta' subunit. Atomic fluorescence spectroscopy showed that prolonged dialysis of the metalloenzyme against 0.01 M o-phenanthroline resulted in the removal of both zinc(II) ions with accompanying loss of enzymatic activity. The activity of the apoenzyme was observed to be completely restored by readdition of zinc(II) and partially restored by cobalt(II).     

Induction of recA protein in Escherichia coli by three platinum (II) compounds

The Na⁺ channels of Chinese Hamster lung fibroblasts have receptor sites for tetrodotoxin, batrachotoxin, veratridine, dihydrograyanotoxin, scorpion and sea anemone toxins. The binding properties of these toxic compounds were determined and shown to be very similar to those found in a variety of excitable cells. Electrophysiological experiments indicate that these Na⁺ channels cannot be electrically activated unless previously treated by veratridine.