trans/cis (Z/E) photoisomerization of the chromophore of photoactive yellow protein is not a prerequisite for the initiation of the photocycle of this photoreceptor protein

The chromophore of photoactive yellow protein (PYP) (i.e., 4-hydroxycinnamic acid) has been replaced by an analogue with a triple bond, rather than a double bond (by using 4-hydroxyphenylpropiolic acid in the reconstitution, yielding hybrid I) and by a “locked” chromophore (through reconstitution with 7-hydroxycoumarin-3-carboxylic acid, in which a covalent bridge is present across the vinyl bond, resulting in hybrid II). These hybrids absorb maximally at 464 and 443 nm, respectively, which indicates that in both hybrids the deprotonated chromophore does fit into the chromophore-binding pocket. Because the triple bond cannot undergo cis/trans (or E/Z) photoisomerization and because of the presence of the lock across the vinyl double bond in hybrid II, it was predicted that these two hybrids would not be able to photocycle. Surprisingly, both are able. We have demonstrated this ability by making use of transient absorption, low-temperature absorption, and Fourier-transform infrared (FTIR) spectroscopy. Both hybrids, upon photoexcitation, display authentic photocycle signals in terms of a red-shifted intermediate; hybrid I, in addition, goes through a blue-shifted-like intermediate state, with very slow kinetics. We interpret these results as further evidence that rotation of the carbonyl group of the thioester-linked chromophore of PYP, proposed in a previous FTIR study and visualized in recent time-resolved x-ray diffraction experiments, is of critical importance for photoactivation of PYP.

Chromophore structural changes in rhodopsin from nanoseconds to microseconds following pigment photolysis

Rhodopsin is a prototypical G protein-coupled receptor that is activated by photoisomerization of its 11-cis-retinal chromophore. Mutant forms of rhodopsin were prepared in which the carboxylic acid counterion was moved relative to the positively charged chromophore Schiff base. Nanosecond time-resolved laser photolysis measurements of wild-type recombinant rhodopsin and two mutant pigments then were used to determine reaction schemes and spectra of their early photolysis intermediates. These results, together with linear dichroism data, yielded detailed structural information concerning chromophore movements during the first microsecond after photolysis. These chromophore structural changes provide a basis for understanding the relative movement of rhodopsin’s transmembrane helices 3 and 6 required for activation of rhodopsin. Thus, early structural changes following isomerization of retinal are linked to the activation of this G protein-coupled receptor. Such rapid structural changes lie at the heart of the pharmacologically important signal transduction mechanisms in a large variety of receptors, which use extrinsic activators, but are impossible to study in receptors using diffusible agonist ligands.

Carboxyfullerenes as neuroprotective agents

Two regioisomers with C3 or D3 symmetry of water-soluble carboxylic acid C60 derivatives, containing three malonic acid groups per molecule, were synthesized and found to be equipotent free radical scavengers in solution as assessed by EPR analysis. Both compounds also inhibited the excitotoxic death of cultured cortical neurons induced by exposure to N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or oxygen-glucose deprivation, but the C3 regioisomer was more effective than the D3 regioisomer, possibly reflecting its polar nature and attendant greater ability to enter lipid membranes. At 100 μM, the C3 derivative fully blocked even rapidly triggered, NMDA receptor-mediated toxicity, a form of toxicity with limited sensitivity to all other classes of free radical scavengers we have tested. The C3 derivative also reduced apoptotic neuronal death induced by either serum deprivation or exposure to Aβ1–42 protein. Furthermore, continuous infusion of the C3 derivative in a transgenic mouse carrying the human mutant (G93A) superoxide dismutase gene responsible for a form of familial amyotrophic lateral sclerosis, delayed both death and functional deterioration. These data suggest that polar carboxylic acid C60 derivatives may have attractive therapeutic properties in several acute or chronic neurodegenerative diseases.

Structural basis for efficient phosphorylation of 3′-azidothymidine monophosphate by Escherichia coli thymidylate kinase

The crystal structures of Escherichia coli thymidylate kinase (TmpK) in complex with P1-(5′-adenosyl)-P5-(5′-thymidyl)pentaphosphate and P1-(5′-adenosyl)P5-[5′-(3′-azido-3′-deoxythymidine)] pentaphosphate have been solved to 2.0-Å and 2.2-Å resolution, respectively. The overall structure of the bacterial TmpK is very similar to that of yeast TmpK. In contrast to the human and yeast TmpKs, which phosphorylate 3′-azido-3′-deoxythymidine 5′-monophosphate (AZT-MP) at a 200-fold reduced turnover number (kcat) in comparison to the physiological substrate dTMP, reduction of kcat is only 2-fold for the bacterial enzyme. The different kinetic properties toward AZT-MP between the eukaryotic TmpKs and E. coli TmpK can be rationalized by the different ways in which these enzymes stabilize the presumed transition state and the different manner in which a carboxylic acid side chain in the P loop interacts with the deoxyribose of the monophosphate. Yeast TmpK interacts with the 3′-hydroxyl of dTMP through Asp-14 of the P loop in a bidentate manner: binding of AZT-MP results in a shift of the P loop to accommodate the larger substituent. In E. coli TmpK, the corresponding residue is Glu-12, and it interacts in a side-on fashion with the 3′-hydroxyl of dTMP. This different mode of interaction between the P loop carboxylic acid with the 3′ substituent of the monophosphate deoxyribose allows the accommodation of an azido group in the case of the E. coli enzyme without significant P loop movement. In addition, although the yeast enzyme uses Arg-15 (a glycine in E. coli) to stabilize the transition state, E. coli seems to use Arg-153 from a region termed Lid instead. Thus, the binding of AZT-MP to the yeast TmpK results in the shift of a catalytic residue, which is not the case for the bacterial kinase.

Polyazamacrolides from ladybird beetles: Ring-size selective oligomerization

The pupal defensive secretion of the 24-pointed ladybird beetle, Subcoccinella vigintiquatuorpunctata, consists of a mixture of macrocyclic polyamines, dominated by the three dimeric, 30-membered macrocycles 11-13, derived from the two building blocks 11-(2-hydoxyethylamino)-5-tetradecenoic acid (9) and 11-(2-hydoxyethylamino)-5,8-tetradecadienoic acid (10). Smaller amounts of the four possible cyclic trimers of 9 and 10 were also detected, corresponding to 45-membered macrocycles. Structural assignments were based on NMR-spectroscopic investigations and HPLC–MS analyses. In addition, the all-S absolute configuration of the S. vigintiquatuorpunctata macrocycles was determined by comparison of derivatives of the natural material with enantiomerically pure synthetic samples. Comparing this alkaloid mixture with that of the pupal defensive secretion in related ladybird beetle species indicates that the degree of oligomerization of the 2-hydroxyethylamino carboxylic acid building blocks can be carefully controlled by the insects.

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Δ strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4β,14α-dimethyl-4α-carboxy-cholesta-8,24-dien-3β-ol and 4β-methyl-4α-carboxy-cholesta-8,24-dien-3β-ol, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2− groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins.

Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Muconate lactonizing enzyme (MLE), a component of the β-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the “enolase superfamily”) that catalyze the abstraction of the α-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-Å resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Brain 5α-dihydroprogesterone and allopregnanolone synthesis in a mouse model of protracted social isolation

Allopregnanolone (ALLO), is a brain endogenous neurosteroid that binds with high affinity to γ-aminobutyric acid type A (GABAA) receptors and positively modulates the action of GABA at these receptors. Unlike ALLO, 5α-dihydroprogesterone (5α-DHP) binds with high affinity to intracellular progesterone receptors that regulate DNA transcription. To investigate the physiological roles of ALLO and 5α-DHP synthesized in brain, we have adopted a mouse model involving protracted social isolation. In the frontal cortex of mice, socially isolated for 6 weeks, both neurosteroids were decreased by approximately 50%. After administration of (17β)-17-(bis-1-methyl amino carbonyl) androstane-3,5-diene-3-carboxylic acid (SKF105,111), an inhibitor of the enzyme (5α-reductase Type I and II) that converts progesterone into 5α-DHP, the ALLO and 5α-DHP content of frontal cortex of both group-housed and socially isolated mice decreased exponentially to 10%–20% of control values in about 30 min. The fractional rate constants (k h−1) of ALLO and 5α-DHP decline multiplied by the ALLO and 5α-DHP concentrations at any given steady-state estimate the rate of synthesis required to maintain that steady state. After 6 weeks of social isolation, ALLO and 5α-DHP biosynthesis rates were decreased to 30% of the values calculated in group-housed mice. Moreover, in socially isolated mice, the expression of 5α-reductase Type I mRNA and protein was approximately 50% lower than in group-housed mice whereas 3α-hydroxysteroid oxidoreductase mRNA expression was equal in the two groups. Protracted social isolation in mice may provide a model to investigate whether 5α-DHP by a genomic action, and ALLO by a nongenomic mechanism down-regulate the action of drugs acting as agonists, partial agonists, or positive allosteric modulators of the benzodiazepine recognition sites expressed by GABAA receptors.

The Mechanism of Rhythmic Ethylene Production in Sorghum. The Role of Phytochrome B and Simulated Shading1

Mutant sorghum (Sorghum bicolor [L.] Moench) deficient in functional phytochrome B exhibits reduced photoperiodic sensitivity and constitutively expresses a shade-avoidance phenotype. Under relatively bright, high red:far-red light, ethylene production by seedlings of wild-type and phytochrome B-mutant cultivars progresses through cycles in a circadian rhythm; however, the phytochrome B mutant produces ethylene peaks with approximately 10 times the amplitude of the wild type. Time-course northern blots show that the mutant's abundance of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase mRNA SbACO2 is cyclic and is commensurate with ethylene production, and that ACC oxidase activity follows the same pattern. Both SbACO2 abundance and ACC oxidase activity in the wild-type plant are very low under this regimen. ACC levels in the two cultivars did not demonstrate fluctuations coincident with the ethylene produced. Simulated shading caused the wild-type plant to mimic the phenotype of the mutant and to produce high amplitude rhythms of ethylene evolution. The circadian feature of the ethylene cycle is conditionally present in the mutant and absent in the wild-type plant under simulated shading. SbACO2 abundance in both cultivars demonstrates a high-amplitude diurnal cycle under these conditions; however, ACC oxidase activity, although elevated, does not exhibit a clear rhythm correlated with ethylene production. ACC levels in both cultivars show fluctuations corresponding to the ethylene rhythm previously observed. It appears that at least two separate mechanisms may be involved in generating high-amplitude ethylene rhythms in sorghum, one in response to the loss of phytochrome B function and another in response to shading.

Regulation of Soybean Nodulation Independent of Ethylene Signaling1

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag+ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag+ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag+. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.

Arg-302 facilitates deprotonation of Glu-325 in the transport mechanism of the lactose permease from Escherichia coli

A mechanistic model for lactose/H+ symport via the lactose permease of Escherichia coli proposed recently indicates that the permease must be protonated to bind ligand with high affinity. Moreover, in the ground state, the symported H+ is shared between His-322 (helix X) and Glu-269 (helix VIII), whereas Glu-325 (helix X) is charge-paired with Arg-302 (helix IX). Substrate binding at the outer surface induces a conformational change that leads to transfer of the H+ to Glu-325 and reorientation of the binding site to the inner surface. After release of the substrate, Glu-325 is deprotonated on the inside because of rejuxtapositioning with Arg-302. To test the role of Arg-302 in the mechanism, the catalytic properties of mutants Arg-302→Ala and Arg-302→Ser were studied. Both mutants are severely defective in active lactose transport, as well as in efflux or influx down a concentration gradient, translocation modes that involve net H+ movement. In marked contrast, the mutants catalyze equilibrium exchange of lactose and bind ligand with high affinity. These characteristics are remarkably analogous to those of permease mutants with neutral replacements for Glu-325, a residue that plays a direct role in H+ translocation. These observations lend strong support for the argument that Arg-302 interacts with Glu-325 to facilitate deprotonation of the carboxylic acid during turnover.

Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G1 and represses expression of CLN1 and CLN2. Mutants with reduced G1 cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G1 arrest upon AZC treatment and temperature up-shift. Finally, we find that the EXA3–1 mutation, encoding a defective HSF, prevents efficient G1 arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G1 arrest.

Biological Activity of Reducing-End-Derivatized Oligogalacturonides in Tobacco Tissue Cultures1

The biological activity of reducing-end-modified oligogalacturonides was quantified in four tobacco (Nicotiana tabacum) tissue culture bioassays. The derivatives used were oligogalacturonides with the C-1 of their reducing end (a) covalently linked to a biotin hydrazide, (b) covalently linked to tyramine, (c) chemically reduced to a primary alcohol, or (d) enzymatically oxidized to a carboxylic acid. These derivatives were tested for their ability to (a) alter morphogenesis of N. tabacum cv Samsun thin cell-layer explants, (b) elicit extracellular alkalinization by suspension-cultured cv Samsun cells, (c) elicit extracellular alkalinization by suspension-cultured N. tabacum cv Xanthi cells, and (d) elicit H2O2 accumulation in the cv Xanthi cells. In all four bioassays, each of the derivatives had reduced biological activity compared with the corresponding underivatized oligogalacturonides, demonstrating that the reducing end is a key element for the recognition of oligogalacturonides in these systems. However, the degree of reduction in biological activity depends on the tissue culture system used and on the nature of the specific reducing-end modification. These results suggest that oligogalacturonides are perceived differently in each tissue culture system.