Andrew J. Baron

The influence of ligand valency on aggregation mechanisms for inhibiting bacterial toxins.

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat‐labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space‐filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head‐to‐head dimerization of the protein toxin en route to higher aggregates.

Affinity of molecular interactions in the bacteriophage φ29 DNA packaging motor

DNA packaging in the bacteriophage φ29 involves a molecular motor with protein and RNA components, including interactions between the viral connector protein and molecules of pRNA, both of which form multimeric complexes. Data are presented to demonstrate the higher order assembly of pRNA together with the affinity of pRNA:pRNA and pRNA:connector interactions, which are used to propose a model for motor function. In solution, pRNA can form dimeric and trimeric multimers in a magnesium-dependent manner, with dissociation constants for multimerization in the micromolar range. pRNA:connector binding is also facilitated by the presence of magnesium ions, with a nanomolar apparent dissociation constant for the interaction. From studies with a mutant pRNA, it appears that multimerization of pRNA is not essential for connector binding and it is likely that connector protein is involved in the stabilization of higher order RNA multimers. It is proposed that magnesium ions may promote conformational change that facilitate pRNA:connector interactions, essential for motor function.

Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins.

The commercial surface plasmon resonance (SPR) biosensors, BIACORE, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators. For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed. The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR. This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription. Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced. This may well be a general mechanism allowing rapid transcription initiation to occur as soon as the repression complex dissociates. These techniques have also been used to examine protein-DNA interactions in the E. coli and Bacillus subtilis arginine repressor systems. The repressors are the products of the argR and ahrC genes, respectively. Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers. There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B. subtilis only). Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine.

Structure and mechanism of galactose oxidase: catalytic role of tyrosine 495

Abstract The catalytic mechanism of the copper-containing enzyme galactose oxidase involves a protein radical on Tyr272, one of the equatorial copper ligands. The first step in this mechanism has been proposed to be the abstraction of a proton from the alcohol substrate by Tyr495, the axial copper ligand that is weakly co-ordinated to copper. In this study we have generated and studied the properties of a Y495F variant to test this proposal. X-ray crystallography reveals essentially no change from wild-type other than loss of the tyrosyl hydroxyl group. Visible spectroscopy indicates a significant change in the oxidised Y495F compared to wild-type with loss of a broad 810-nm peak, supporting the suggestion that this feature is due to inter-ligand charge transfer via the copper. The presence of a peak at 420 nm indicates that the Y495F variant remains capable of radical formation, a fact supported by EPR measurements. Thus the significantly reduced catalytic efficiency (1100-fold lower kcat / Km) observed for this variant is not due to an inability to generate the Tyr272 radical. By studying azide-induced pH changes, it is clear that the reduced catalytic efficiency is due mainly to the inability of Y495F to accept protons. This provides definitive evidence for the key role of Tyr495 in the initial proton abstraction step of the galactose oxidase catalytic mechanism.

The Klebsiella aerogenes glutamate dehydrogenase (gdhA) gene: cloning, high-level expression and hybrid enzyme formation in Escherichia coli

SummaryThe NADP-dependent glutamate dehydrogenase gene of Klebsiella aerogenes was cloned in E. coli in the expression plasmid pRK9. The cloned gene shows a high level of expression in E. coli in the hybrid plasmid pKG3 and such expression is independent of the vector promoter, as shown by experiments in which the promoter was deleted. Active hybrid GDH hexamers were shown in cell-free extracts of an E. coli strain carrying cloned gdhA genes of both E. coli and K. aerogenes. The nucleotide sequence of the N-terminal coding region of the K. aerogenes gdhA gene was determined and found to be strongly homologous with that of E. coli.

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes : gene cloning, sequence analysis and high-level production in Escherichia coli

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase: interactions of the GOasesemi state with different buffers, its redox activity and ability to bind azide

Abstract The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ∼8 Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400 mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25  °C, I=0.100 M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOasesemi (CuII) form of the Trp290His variant. Equilibrium constants Keq/M–1 from absorbance changes at 635 nm are for 1 : 1 interactions with the OH-containing buffers H2PO4– (231), Hepes (43) and Tris (202), concentrations 0–60 mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ∼635 nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOaseox/GOasesemi couple is 730 mV, which compares with 400 mV for the WT GOase couple. Consistent with the 730 mV value the GOasesemi form is not oxidised with [Fe(CN)6]3– (410 mV) or [W(CN)8]3– (530 mV), and much stronger oxidants such as [Mo(CN)8]3– (800 mV) and [IrCl6]2– (890 mV) are required. The GOaseox product is unstable and decays within 20 min with re-formation of GOasesemi. From changes in UV-Vis spectra with pH, Trp290His GOasesemi gives a pKa of 6.9, and rate constants for the oxidation of GOasesemi with [Mo(CN)8]3– are dependent on this same pKa. The latter compares with 7.9 for WT GOasesemi, and is assigned here also as protonation of Tyr-495. The 1 : 1 binding of azide at the substrate-binding (H2O) site of Trp290His GOasesemi was studied and gives a formation constant 330 M–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOasesemi. The trends observed indicate less affinity of Trp290His GOasesemi for the ionic reactants H+ and N3–.

Co‐translational myristoylation alters the quaternary structure of HIV‐1 Nef in solution

We have studied the solution properties of Nef, a 24‐kDa cotranslationally myristoylated protein produced by HIV‐1 and other primate lentiviruses. Nef is found in the cytosol and also in association with cytoplasmic membranes, the latter, mediated in part by the myristoyl group attached to the N‐terminal glycine. Recombinant Nef was coexpressed in Escherichia coli in tandem with N‐myristoyl‐transferase and is fully myristoylated. Analysis by circular dichroism showed the myristoylated form to contain a greater α‐helical content than the nonmyristoylated form. Analysis of modified and unmodified Nef in solution using small angle X‐ray scattering, dynamic laser light scattering and analytical ultracentrifugation consistently showed differences in the oligomeric states of the two forms of Nef. Myristoylated Nef is predominantly monomeric and small oligomers which are also present, can be converted to the monomeric form under reducing conditions. By contrast, the nonmyristoylated form exists as a stable hexadecamer in solution which disassociates into tetramers upon addition of reducing agents. Shape reconstructions from small angle scattering curves of nonmyristoylated Nef are compatible with a large disc‐like structure in the hexadecameric oligomer consisting of four Nef tetramers. From these findings, we hypothesize that Nef undergoes a substantial conformational change from an “open” into a “closed” form whereby the myristate group is sequestered in a hydrophobic pocket. The myristoylated protein can switch to the open conformation by association of the N‐terminal region of molecule with membranes. These changes would allow Nef to carry out various functions depending on the conformational and oligomeric states. Proteins 2005.

Respiratory nitrate reductase of Escherichia coli. Sequence identification of the large subunit gene

Nitrate reductase Nar operon DNA sequence Amino acid sequence Molybdoprotein (Escherichia coli)

Characterization of the active site of galactose oxidase and its active site mutational variants Y495F/H/K and W290H by circular dichroism spectroscopy

Circular dichroism spectroscopy (CD) has been used to investigate the generation of the tyrosine radical in wild-type galactose oxidase and the active site variants Y495F/H/K and W290H. Oxidation was observed in all the variants except Y495K and the radical was noted to have a greater stability at pH 4.6 compared to pH 7.0, especially in Y495H and W290H. In the axial tyrosine variants active site oxidation to generate the radical species was confirmed by the presence of characteristic CD bands, particularly a negative band, in the 350 to 450 nm region. The band at 810 nm in the optical absorption spectrum of WT-GO is absent in oxidized Y495 variants consistent with the Y495 → Y272 via Cu(II)dA, assignment (M.L. McGlashen, D.D. Eads, T.G. Spiro and J.W. Whittaker, J. Phys. Chem., 99 (1995) 4918–4922 [1]). CD spectra of either oxidized or semi-reduced proteins are pH-dependent between pH 4.6 and 7.0 with differing intersities and dispersions. The presence of a positive CD band between 309 and 321 nm (N(π) → Cu(II)) confirmed the coordination of histidine to the copper ion in the variants studied here. The slight wavelength and intensity shifts seen in this transition is ascribed to perturbation of coupling of the dyssymmetric environment to the electronic transitions of the copper site.