Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142–156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions—all of which could influence the final conformation of the enzyme.     

Helix packing of leucine-rich peptides: a parallel leucine ladder in the structure of Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe.

The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A.     

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli

Summary The effects of 25-fold overproduction ofEscherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from theBacillus subtilis chromosome and the mature part of TEM-β-lactamase, were studied inE. coli. One precursor (pre[A2d]-β-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[Al3i]-β-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature β-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-β-lactamase and pre(A2)-β-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export inE. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.     

Horse heart ferricytochrome c: conformation and heme configuration of high ionic strength acidic forms.

The absorption, circular dichroism, and resonance Raman spectra of horse heart ferricytochromec in the presence of 0.2 M KCl, 0.1 M NaClO₄, and 0.2 M KNO₃, in thepH region 7 to 0.5, have been investigated to determine the nature and the course of the processes involved. As in the absence of salts (Myer, Y., and Saturno, A. F. (1990)J. Protein Chem.,9, 379–387), the change from neutral to low acidicpH's in the presence of salts is a three-step process: state III _s state III _s,a state II _s state I _s , withpK _a 's of 3.5±0.2, 2.2±0.2, and 1.1±0.2, and with two, one, and one number of protons, respectively. The addition of salts at neutralpH's has little or no effect on the protein conformation and the heme-iron configuration (i.e., they remain the same, low-spin hexacoordinated heme iron with a Met-80-Fe-His-18 axial coordination), but such addition does cause a slight tightening of the heme crevice and the enlargement of the porphyrin core. State III _s,a is a folded state with about the same degree of folding and with a similar spin state and coordination configuration of iron, but the heme crevice is loosened and the porphyrin core is smaller. Both states II _s and I _s are also essentially folded forms, but with a smaller degree of protein secondary structure. State II _s has a high-spin hexacoordinated heme iron with a water molecule and a protonated and/or hydrogen-bonded imidazole of his-18 as the two axial ligates; and state I _s has a high-spin pentacoordinated heme iron, which is about 0.49 Å out of the porphyrin plane, with a protonated and/or hydrogen-bonded imidazole nitrogen as the only axial ligate. The addition of anions causes the stabilization of the protein secondary structures and the state III _a state II transition. The mode of effectiveness of anions appears to be nonspecific (i.e., because of electrostatic shielding and/or disruption of salt bridges).     

Backbone side-chain interactions in peptides

IR, ¹H-NMR and X-ray experiments have been carried out on dipeptides with the Pro-Asp and Pro-Asn sequences protected on both ends by amide groups. The Pro-Asp dipeptide was investigated for the carboxylic, methyl ester and carboxylate forms of the Asp residue.In solution, all dipeptides are found to accommodate almost exclusively the I-turn conformation stabilized by an interaction between the Asp or Asn-NH and CO bonds. The I-turn percentage roughly parallels the basicity of the Asp or Asn side substituent, and decreases from Asp^- to Asn, and to Asp or Asp (OMe).The I-turn, stabilized by the interaction involving the Asp-CO site, is retained in the crystal structure of the Pro-Asp(OMe) dipeptide. The Pro-Asp and Pro-Asn dipeptides assume a II-turn conformation in the solid state and the polar Asp or Asn side-groups are involved in a complex network of intermolecular interactions.     

Evidence for two distinct conformations of the Escherichia coli mannitol permease that are important for its transport and phosphorylation functions

Column chromatography of the Escherichia coli mannitol permease (mannitol-specific enzyme II of the phosphotransferase system) in the presence of deoxycholate has revealed that the active permease can exist in at least two association states with apparent molecular weights consistent with a monomer and a dimer. The monomeric conformation is favored by the presence of mannitol and by the phosphoenolpyruvate (PEP)-dependent phosphorylation of the protein. The dimer is stabilized by inorganic phosphate (Pi), which also stimulates phospho-exchange between mannitol and mannitol 1-phosphate (a partial reaction in the overall PEP-dependent phosphorylation of mannitol). Kinetic analysis of the phospho-exchange reaction revealed that Pi stimulates phospho-exchange by increasing the Vmax of the reaction. A kinetic model for mannitol permease function is presented involving both conformations of the permease. The monomer (or a less-stable conformation of the dimer) is hypothesized to be involved in the initial mannitol-binding and PEP-dependent phosphorylation steps, while the stably associated dimer is suggested to participate in later steps involving direct phosphotransfer between the permease, mannitol and mannitol 1-phosphate.     

Induction of ?2 frameshift mutations within alternating GC sequences by carcinogens that bind to the C8 position of guanine residues Development of a specific mutation assay

Summary Using a forward mutation assay we have previously found that N-2-acetylaminofluorene (AAF), a strong chemical carcinogen, induces a majority of frameshift mutations located at specific sequences called mutation hot spots. Among these hot spot sequences, the NarI sequence (GGCGCC), is specific for –2 frameshifts (GGCGCC) GGCC). Interestingly, these frameshift mutations occur independently of a functional umuDC locus. Being interested in elucidating this mutation pathway we have developed a reversion assay that is specific for this class of mutations. The assay is based on the reversion of a +2 frameshift mutant of plasmid pBR322 from tetracycline sensitivity to tetracycline resistance. It is shown that only true reversion events lead to tetracycline resistance. The carcinogen AAF induces this reversion event at a frequency that is increased four- to fivefold over the background frequency. A series of chemical carcinogens which, like AAF, bind covalently to the C8 position of guanine, are compared for their efficiency to induce this specific mutation event. Large variations in the mutagenic efficiency of these chemicals are observed and discussed in terms of the anti/syn conformation of the carcinogen-modified guanine residue. Based on this test, we describe a convenient spot assay that this presently used in our laboratory to isolate Escherichia coli mutants affected in this mutation pathway.     

Aspects of the demetalation and remetalation of ceruloplasmin

This study dealt with the demetalation and remetalation chemistry of the copper-containing protein ceruloplasmin. For the enzyme from human plasma, dialysis against cyanide at 4°C readily removed copper. Although the apoprotein took up copper(I) at the same temperature, the characteristic blue color of the native protein did not return to any significant extent. However, excellent reconstitution occurred when we added copper(I) at room temperature. With porcine ceruloplasmin, it was more practical to carry out the copper removal step at room temperature, but the reconstitution went smoothly at 4°C. With either source of ceruloplasmin, the binding of the six essential copper ions was generally a highly cooperative process, but the results were different when we combined the apoprotein with Hg(II). After the protein took up 2 mercury ions, it would take up only 1–2 more metal ions even after exposure to a large excess of copper(I). In order to accomodate the various experimental results, we have proposed that a reversible conformational change must occur during the demetalation and remetalation processes. During the remetalation process, it is therefore important that metal uptake occurs in the proper sequence.     

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at Gly-149 was suppressed by the second mutations at the outer surface of the subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that / and / subunits interactions are important for the energy coupling between catalysis and H⁺ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

Conformation of 4-thio-l-lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio-l-lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.