The rnf genes of Rhodobacter capsulatus, essential for nitrogen fixation, are thought to encode a system for electron transport to nitrogenase. In the present study, we have attempted to overexpress the rnf genes in Escherichia coli to investigate the molecular properties of the corresponding proteins. Corrections were made to the published DNA sequence of the rnf operon, resulting in the identification of two genes, rnfG and rnfH. The rnfABCDGEH operon thus comprises seven genes and shows similarities in gene arrangement and deduced protein sequences to homologous regions in the genomes of Haemophilus influenzae and E. coli. Four of the rnf gene products were found to be similar in sequence to components of an Na ϩ -dependent NADH:ubiquinone oxidoreductase from Vibrio alginolyticus. Three of the rnf genes were successfully overexpressed in E. coli as His-tagged polypeptides, whereas the products of rnfA, rnfD and rnfE, predicted to be transmembrane proteins, could not be stably maintained in E. coli. The rnfB and rnfC gene products were isolated as two brown proteins with apparent molecular-mass values of 25 kDa and 55 kDa, respectively. RnfB was shown to contain one [2Fe-2S] cluster, based on absorption spectrophotometry, EPR spectroscopy and iron content. Recombinant RnfC contained at least one ironsulfur cluster, most likely of the [4Fe-4S] type. Unambiguous identification of the prosthetic groups was, however, precluded by the extreme instability of this protein. In R. capsulatus, RnfB and RnfC were found by immunoblot analysis to be tightly bound to the membrane, despite their hydrophilic character. The RnfB and RnfC proteins were absent in mutant strains bearing insertions at various positions within the rnfABCDGEH operon, suggesting that their stability depends on the cosynthesis of the other rnf gene products. We observed that iron limitation during growth resulted in a decrease both in the cellular content of RnfB and in the level of transcription of the rnfABCDGEH operon, indicating that the expression of this operon is regulated as a function of iron availability

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA.

Catechol 2,3-dioxygenase (XylE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygensensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE. In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyperexpressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin. It is the first example of a soluble ferredoxin with a net positive charge at neutral pH. The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin. The midpoint redox potential was determined to be ؊373 ؎ 6 mV, at pH 8.5. XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25°C in air and 70 min at 37°C in argon. With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro. Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR. Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.Pseudomonas putida mt2 carries the TOL plasmid pWW0, which encodes a set of enzymes responsible for the transformation of toluene and xylene to central pathway intermediates (1).The catabolic (xyl) genes are organized in two operons, the so-called upper and meta-operons. The enzymes encoded by the upper operon catalyze the sequential oxidation of toluene to benzoate, whereas the enzymes of the meta-operon convert benzoate to Krebs cycle intermediates (2). In the meta-pathway, the enzyme catechol 2,3-dioxygenase encoded by xylE catalyzes the extradiol cleavage of the aromatic ring. This reaction has been studied in detail, and a general mechanism for the oxidative cleavage of catechol by extradiol dioxygenases has been proposed (3). The two atoms of the oxygen molecule are incorporated in the catechol substrate on two adjacent carbon atoms of the aromatic ring, one of which already carries a hydroxyl substituent of the diol and the other of which is unsubstituted. At the active site, the enzyme possesses a single iron atom that binds the substrate and oxygen and participates in the catalytic cycle.

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The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were ؊286 mV and ؊323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M r of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenaseferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.In bacteria that are able to perform oxidative catabolism of aromatic hydrocarbons, degradation pathways converge towards a few common intermediates such as protocatechuate, salicylate, hydroxyquinol, and catechol (7). These intermediates are further oxidized by dioxygenases, which cleave the aromatic ring. Enzymatic cleavage of catechol is catalyzed by two categories of catechol dioxygenases, referred to as intradiol and extradiol dioxygenases, depending on the position of the cleavage site relative to the diol. The two types of enzymes are biochemically and structurally different and have distinct phylogenetic origins (9). The catechol 2,3-dioxygenase from Pseudomonas putida mt2, an archetypical extradiol dioxygenase, has been extensively studied, and its crystal structure has recently been solved (20). The enzyme is a tetramer composed of four identical subunits, each of which contains a ferrous iron atom. It is a labile enzyme which is inactivated upon exposure to oxidizing agents such as oxygen (24). Inactivation also occurs during catalytic turnover wh...

A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25°C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.Key words: Co-variance; antibody surface charges; Biacore; antigen-binding properties; half-life; recombinant protein yield A mutational analysis of a recombinant antibody fragment was performed with two aims: (1) to gain a better understanding of the relationship between antibody sequence and function and (2) to identify candidate positions in engineering antibodies for biotechnological or medical applications.We are searching for protein engineering rules, applicable to at least a subgroup of antibodies, and have selected for mutagenesis residues located within the structurally conserved framework region, rather than in the highly variable complementarity determining regions (CDRs) that determine antigen-binding specificity. The residues in question are co-variant positions that were identified in an analysis of antibody germline sequence alignments : Two positions are co-variant if the nature of amino acids at position A is not independent of the nature of amino acids at position B, indicating the existence of functional constraints linking these two residues (Altschuh et al. 1987). The study by Choulier et al. (2000) indicated the existence of a co-variance signal for framework positions located at the surface of the variable region. In particular, for two co-variant pairs identified in mouse antibody germline sequences (L18-L74, H46-H62, Kabat numbering), alternative amino acid types presented large differences in solvation free energ...

Predictive engineering of antibodies exhibiting fast kinetic properties could provide reagents for biotechnological applications such as continuous monitoring of compounds or affinity chromatography. Based on covariance analysis of murine germline antibody variable domains, we selected position L34 (Kabat numbering) for mutational studies. This position is located at the VL/VH interface, at the base of the paratope but with limited antigen contacts, thus making it an attractive position for mild alterations of antigen binding properties. We introduced a serine at position L34 in two different antibodies: Fab (fragment antigen binding) 57P (Asn34Ser) and scFv (single chain fragment variable) 1F4 (Gln34Ser), that recognize peptides derived from the coat protein of tobacco mosaic virus and the oncoprotein E6, respectively. Both mutated antibodies exhibited similar properties: (i) expression levels of active fragments in Escherichia coli were markedly improved; (ii) thermostability was enhanced; and (iii) dissociation rate parameters (k(off)) were increased by 2- and at least 57-fold for scFv1F4 and Fab57P, respectively, while their association rate parameters (k(on)) remained unchanged. The L34 Ala and Thr mutants of both antibody fragments did not possess these properties. This first demontration of similar effects observed in two antibodies with different specificities may open the way to the predictive design of molecules with enhanced stability and fast dissociation rates.

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