Structural investigation of oxidized chlorosomes from green bacteria using multifrequency electron paramagnetic resonance up to 330 GHz

Chemical oxidation of the chlorosomes from Chloroflexus aurantiacus and Chlorobium tepidum green bacteria produces bacteriochlorophyll radicals, which are characterized by an anomalously narrow EPR signal compared to in vitro monomeric BChl c ^.+ [Van Noort PI, Zhu Y, LoBrutto R and Blankenship RE (1997) Biophys J 72: 316–325]. We have performed oxidant concentration and temperature-dependent X-band EPR measurements in order to elucidate the line narrowing mechanism. The linewidth decreases as the oxidant concentration is increased only for Chloroflexus indicating that for this system Heisenberg spin exchange is at least partially responsible for the EPR spectra narrowing. For both species the linewidth is decreasing on increasing the temperature. This indicates that temperature-activated electron transfer is the main narrowing mechanism for BChl radicals in chlorosomes. The extent of the electron transfer process among different BChl molecules has been evaluated and a comparison between the two species representative of the two green bacteria families has been made. In parallel, high frequency EPR experiments have been performed on the oxidized chlorosomes of Chloroflexus and Chlorobium at 110 and 330 GHz in the full temperature range investigated at X-band. The g-tensor components obtained from the simulation of the 330 GHz EPR spectrum from Chlorobium show the same anisotropy as those of monomeric Chl a ^.+ [Bratt PJ, Poluektov OG, Thurnauer MC, Krzystek J, Brunel LC, Schrier J, Hsiao YW, Zerner M and Angerhofer A (2000) J Phys Chem B 104: 6973–6977]. The spectrum of Chloroflexus has a nearly axial g-tensor with reduced anisotropy compared to Chlorobium and monomeric Chl a in vitro. g-tensor values and temperature dependence of the linewidth have been discussed in terms of the differences in the local structure of the chlorosomes of the two families.This revised version was published online in October 2005 with corrections to the Cover Date.     

The synthesis and characterization of BMS-204352 (MaxiPost) and related 3-fluorooxindoles as openers of maxi-K potassium channels

3-Aryl-3-fluorooxindoles can be efficiently synthesized in two steps by the addition of an aryl Grignard to an isatin, followed by treatment with DAST. Oxindole 1 (BMS-204352; MaxiPost) can be isolated using chiral HPLC or prepared by employing chiral resolution. Cloned maxi-K channels are opened by 1, which demonstrates a brain/plasma ratio >9 in rats.     

EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts

We have established transgenic mice over-expressing the EphB4 receptor tyrosine kinase in the kidney. The EphB4 protein was localised to the developing tubular system of both control and transgenic newborn mice. In transgenic adults, transgene expression persisted in the proximal tubules and the Bowman's capsules, structures, which were not stained in control kidneys. The glomeruli of control animals consisted of regular, round vascular baskets with clearly discernable afferent and efferent arterioles. In contrast, approximately 40% of the transgenic glomeruli had an irregular shrivelled appearance and many exhibited fused, horse shoe-like afferent and efferent arterioles bypassing the glomerulus. These abnormal glomerular structures are very reminiscent of aglomerular vascular shunts, a human degenerative glomerulopathy of unknown aetiology.     

Formation and functioning of fused cholesterol side-chain cleavage enzymes

We studied the properties of various fused combinations of the components of the mitochondrial cholesterol side-chain cleavage system including cytochrome P450scc, adrenodoxin (Adx), and adrenodoxin reductase (AdR). When recombinant DNAs encoding these constructs were expressed in Escherichia coli, both cholesterol side-chain cleavage activity and sensitivity to intracellular proteolysis of the three-component fusions depended on the species of origin and the arrangement of the constituents. To understand the assembly of the catalytic domains in the fused molecules, we analyzed the catalytic properties of three two-component fusions: P450scc-Adx, Adx-P450scc, and AdR-Adx. We examined the ability of each fusion to carry out the side-chain cleavage reaction in the presence of the corresponding missing component of the whole system and examined the dependence of this reaction on the presence of exogenously added individual components of the double fusions. This analysis indicated that the active centers in the double fusions are either unable to interact or are misfolded; in some cases they were inaccessible to exogenous partners. Our data suggest that when fusion proteins containing P450scc, Adx, and AdR undergo protein folding, the corresponding catalytic domains are not formed independently of each other. Thus, the correct folding and catalytic activity of each domain is determined interactively and not independently.     

Targeted disruption of the mouse Mel(1b) melatonin receptor

Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel(1a) melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel(1b) melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel(1b) receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel(1b) receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel(1a) receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel(1a) receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel(1a)-Mel(1b) double-mutant mice, indicating that the Mel(1b) receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.     

Sequence gamma 377-395(P2), but not gamma 190-202(P1), is the binding site for the alpha MI-domain of integrin alpha M beta 2 in the gamma C-domain of fibrinogen

The interaction between the leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1, CR3) and fibrinogen mediates the recruitment of phagocytes during the inflammatory response. Previous studies demonstrated that peptides P2 and P1, duplicating gamma 377-395 and gamma 190-202 sequences in the gamma C domain of fibrinogen, respectively, blocked the fibrinogen-binding function of alpha(M)beta(2), implicating these sequences as possible binding sites for alpha(M)beta(2). To determine the role of these sequences in integrin binding, recombinant wild-type and mutant gamma C domains were prepared, and their interactions with the alpha(M)I-domain, a ligand recognition domain within alpha(M)beta(2), were tested. Deletion of gamma 383-411 (P2-C) and gamma 377-411 produced gamma C mutants which were defective in binding to the alpha(M)I-domain. In contrast, alanine mutations of several residues in P1 did not affect alpha(M)I-domain binding, and simultaneous mutations in P1 and deletion of P2 did not decrease the binding function of gamma C further. Verifying the significance of P2, inserting P2-C and the entire P2 into the homologous position of the beta C-domain of fibrinogen imparted the higher alpha(M)I-domain binding ability to the chimeric proteins. To further define the molecular requirements for the P2-C activity, synthetic peptides derived from P2-C and a peptide array covering P2-C have been analyzed, and a minimal recognition motif was localized to gamma(390)NRLTIG(395). Confirming a critical role of this sequence, the cyclic peptide NRLTIG retained full activity inherent to P2-C, with Arg and Leu being important residues. Thus, these data demonstrate the essential role of the P2, but not P1, sequence for binding of gamma C by the alpha(M)I-domain and suggest that the adhesive function of P2 depends on the minimal recognition motif NRLTIG.     

The role of alternative splicing and C-terminal amino acids in thromboxane receptor stabilization

The thromboxane receptor has two alternatively spliced isoforms, alpha and beta, which differ only in sequences within the cytoplasmic C-terminal domain. Oxidative stress induced by H(2)O(2) in a COS-7 cell model results in stabilization of the thromboxane receptor beta isoform by translocation from the endoplasmic reticulum to the Golgi complex, which in turn results in protection of the receptor from degradation. We now report that both the alpha and beta thromboxane receptor isoforms respond identically to oxidative stress. Further, mutagenesis studies indicate that replacing the normal C-terminus with a nonsense sequence also does not alter stabilization behaviour ruling out a role for the distinct C-termini in this process. Further mutagenesis implicates a cluster of arginine residues within the C-terminal domain as involved in oxidative stress-induced stabilization. These data identify a region of the thromboxane receptor that is responsible for responding to oxidative challenge and open the possibility of identification of the molecular machinery underpinning this response.     

Sequence-specific artificial ribonucleases. I. Bis-imidazole-containing oligonucleotide conjugates prepared using precursor-based strategy

Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5′-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNA^Phe (44–60 nt) and was expected to deliver imidazole groups near the fragile sequence C₆₁-ACA-G₆₅ of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C₆₃–A₆₄ phosphodiester bond. For some constructs, cleavage was completed within 1–2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the ‘active’ cleaving conformation(s).     

Hybridization of antisense oligonucleotides with alpha-sarcin loop region of Escherichia coli 23S rRNA

Binding of complementary oligonucleotides (ONs) with alpha-sarcin loop region (2638-2682) of Escherichia coli 23S rRNA was investigated. Four of the tested pentadecanucleotides efficiently bound to target sequences with association rate and equilibrium constants approximately 10(3) M(-1)s(-1) and 10(7) M(-1), respectively. ON S5 (CGAGAGGACCGGAGU) complementary to the sequence 2658-2672 displayed the highest affinity to the target. Activation energy for binding of ON S5 was measured to be 11 kcal/mol; this value corresponds to approximately 10% of the calculated enthalpy of the local RNA structure unfolding in the presence of this oligonucleotide. The activation energy value is evidence for the heteroduplex formation to occur via strand displacement pathway; the initiation of heteroduplex formation requires disruption of 1-2 base pairs in RNA hairpin.     

Electron transfer in crystals of the binary and ternary complexes of methylamine dehydrogenase with amicyanin and cytochrome<Emphasis Type="Italic"> c</Emphasis>551i as detected by EPR spectroscopy

EPR studies of the methylamine dehydrogenase (MADH)–amicyanin and MADH–amicyanin–cytochrome c551i crystalline complexes have been performed on randomly oriented microcrystals before and after exposure to the substrate, methylamine, as a function of pH. The results show that EPR signals from the redox centers present in the various proteins can be observed simultaneously. These results complement and extend earlier studies of the complexes under similar conditions that utilized single-crystal polarized absorption microspectrophotometry. The binary complex shows a blue copper axial signal, characteristic of oxidized amicyanin. After reaction of substrate with the MADH coenzyme tryptophan tryptophylquinone (TTQ), the binary complex exhibits an equilibrium mixture of oxidized copper/reduced TTQ and reduced copper/TTQ· radical, whose ratio is dependent on the pH. In the oxidized ternary complex, the same copper axial signal is observed superimposed on the low-spin ferric heme features characteristic of oxidized cytochrome c551i. After addition of substrate to the ternary complex, a decrease of the copper signal is observed, concomitant with the appearance of the radical signal derived from the semiquinone form of TTQ. The equilibrium distribution of electrons between TTQ and copper as a function of pH is similar to that observed for the binary complex. This result was essential to establish that the copper center retains its function within the crystalline ternary complex. At high pH, with time the low-spin heme EPR features disappear and the spectrum indicates that full reduction of the complex by substrate has occurred.