A synergistic effect of phosphate, pH and Phe159 substitution on the formycin A association to the E. coli purine nucleoside phosphorylase.

A steady-state absorption and emission spectroscopy was used to create a comprehensive work and to study the interaction of the wild type Escherichia coli purine nucleoside phosphorylase and its mutants, PNPF159Y and PNPF159A, with a potent E. coli PNP inhibitor - formycin A. The absorption and emission spectra were recorded in the presence and absence of the phosphate at the 50 mM concentration. From the collected sets of data dissociation constants (Kd), apparent dissociation constants (Kapp) and Hill's coefficients (h) were calculated. Additionally, the temperature dependence of the enzymes emission quenching at two temperatures, 10 °C and 25 °C, was examined. To verify the calculations, total difference absorption spectra were computed for all types of the complexes. A prominent quenching of the PNPF159Y emission indicates a complex formation, with the strongest association in the phosphate buffer, pH 7, relative to the wild type enzyme. On the other hand, results testify to a deterioration of the interactions in the E. coli PNP/PNPF159Y and formycin A complexes in the presence of the phosphate, pH 8.3. Moreover, data obtained for the PNPF159A-FA complexes confirm a weak association of the FA to the mutant's active center.

Towards understanding the E. coli PNP binding mechanism and FRET absence between E. coli PNP and formycin A.

The aim of this study is threefold: (1) augmentation of the knowledge of the E. coli PNP binding mechanism; (2) explanation of the previously observed 'lack of FRET' phenomenon and (3) an introduction of the correction (modified method) for FRET efficiency calculation in the PNP-FA complexes. We present fluorescence studies of the two E. coli PNP mutants (F159Y and F159A) with formycin A (FA), that indicate that the aromatic amino acid is indispensable in the nucleotide binding, additional hydroxyl group at position 159 probably enhances the strength of binding and that the amino acids pair 159-160 has a great impact on the spectroscopic properties of the enzyme. The experiments were carried out in hepes and phosphate buffers, at pH7 and 8.3. Two methods, a conventional and a modified one, that utilizes the dissociation constant, for calculations of the energy transfer efficiency (E) and the acceptor-to-donor distance (r) between FA and the Tyr (energy donor) were employed. Total difference spectra were calculated for emission spectra (λex 280nm, 295nm, 305nm and 313nm) for all studied systems. Time-resolved techniques allowed to conclude the existence of a specific structure formed by amino acids at positions 159 and 160. The results showed an unexpected pattern change of FRET in the mutants, when compared to the wild type enzyme and a probable presence of a structure created between 159 and 160 residue, that might influence the binding efficiency. Additionally, we confirmed the indispensable role of the modification of the FRET efficiency (E) calculation on the fraction of enzyme saturation in PNP-FA systems.

Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N(4)-hydroxy-dCMP.

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N(4)-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N(5,10)-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N(4)-hydroxy-dCMP was found to be strongly dependent on [Mg(2+)], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.

[Role of tautomerism in the molecular mechanisms of mutagenesis]. / Rola tautomerii w molekularnych mechanizmach mutagenezy.

Environment of human being usually contains a high number of environmental mutagens, which may modify chemically nucleic acid bases into promutagenic analogues. Hydroxylamine (NH2OH) is a strong mutagen which modifies cytosine and adenine to N4-hydroxycytosine and N6-hydroxyadenine, respectively. Once these analogues are present in DNA or RNA, they may cause transition point mutations by the exchange between two pairs C:G and A:T into T:A and G:C, respectively. The reason for these mutations is the change of preferences between intermolecular hydrogen bonds resulting from the shift of the tautomeric equilibrium from the preferred amino form into the imino one. In the case of the amino<-->imino tautomeric equilibrium of N6-hydroxyadenosine, it was also shown that preferential hydrogen bonding between its imino form and cytidine, or uridine and the amino form of this base leads to the shift of the tautomeric equilibrium in favour of these tautomers in solution. N4-hydroxy-dCMP analogues exhibited very interesting inhibitory properties versus the biosynthesis of dTMP catalyzed by thymidylate synthase. These properties help to further the knowledge on the molecular mechanism of the catalytic reaction of this enzyme as well as on the role of syn-anti photoisomerization of the N4-hydroxy group in this reaction. Examinations gathered in the article were conducted from 1979 to 1985 under supervision, and afterwards from 1986 to 2004 in collaboration with professor David Shugar.

Development of novel molecular probes of the Rio1 atypical protein kinase.

The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).

Fluorescence anisotropy of tyrosinate anion using one-, two- and three-photon excitation: tyrosinate anion fluorescence.

We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250-310 nm), two-photon (570-620 nm) and three-photon (750-930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λmax 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565-580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at -60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos(6) θ to cos(2) θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.

Correlation between spatial (3D) structure of pea and bean thylakoid membranes and arrangement of chlorophyll-protein complexes.

BACKGROUND: The thylakoid system in plant chloroplasts is organized into two distinct domains: grana arranged in stacks of appressed membranes and non-appressed membranes consisting of stroma thylakoids and margins of granal stacks. It is argued that the reason for the development of appressed membranes in plants is that their photosynthetic apparatus need to cope with and survive ever-changing environmental conditions. It is not known however, why different plant species have different arrangements of grana within their chloroplasts. It is important to elucidate whether a different arrangement and distribution of appressed and non-appressed thylakoids in chloroplasts are linked with different qualitative and/or quantitative organization of chlorophyll-protein (CP) complexes in the thylakoid membranes and whether this arrangement influences the photosynthetic efficiency. RESULTS: Our results from TEM and in situ CLSM strongly indicate the existence of different arrangements of pea and bean thylakoid membranes. In pea, larger appressed thylakoids are regularly arranged within chloroplasts as uniformly distributed red fluorescent bodies, while irregular appressed thylakoid membranes within bean chloroplasts correspond to smaller and less distinguished fluorescent areas in CLSM images. 3D models of pea chloroplasts show a distinct spatial separation of stacked thylakoids from stromal spaces whereas spatial division of stroma and thylakoid areas in bean chloroplasts are more complex. Structural differences influenced the PSII photochemistry, however without significant changes in photosynthetic efficiency. Qualitative and quantitative analysis of chlorophyll-protein complexes as well as spectroscopic investigations indicated a similar proportion between PSI and PSII core complexes in pea and bean thylakoids, but higher abundance of LHCII antenna in pea ones. Furthermore, distinct differences in size and arrangements of LHCII-PSII and LHCI-PSI supercomplexes between species are suggested. CONCLUSIONS: Based on proteomic and spectroscopic investigations we postulate that the differences in the chloroplast structure between the analyzed species are a consequence of quantitative proportions between the individual CP complexes and its arrangement inside membranes. Such a structure of membranes induced the formation of large stacked domains in pea, or smaller heterogeneous regions in bean thylakoids. Presented 3D models of chloroplasts showed that stacked areas are noticeably irregular with variable thickness, merging with each other and not always parallel to each other.

3-D modelling of chloroplast structure under (Mg2+) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking.

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.

Analysis of proton exchange kinetics with time-dependent exchange rate.

Mass spectrometry is used to probe the kinetics of hydrogen-deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen-deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.

Fluorescence emission properties of 8-aza analogues of caffeine, theophylline, and related N-alkyl xanthines.

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan, is highly fluorescent. All three 8-azaxanthines monomethylated on the triazole ring, as well as 8-azaxanthosine, exhibit increased acidity in the excited state. Some fluorescent pyrazolo[4,3-d]pyrimidine-5,7-diones, xanthine congeners of pyrazolo[4,3-d]pyrimidines, are also reported. Many of these are good fluorescent probes in enzymatic, receptor binding, and nucleic acid systems, some examples of which are presented. In particular, 8-azaXan is an excellent fluorescent probe for purine nucleoside phosphorylases, as a fluorogenic substrate in the reverse, synthetic pathway.

Role of ionization of the phosphate cosubstrate on phosphorolysis by purine nucleoside phosphorylase (PNP) of bacterial (E. coli) and mammalian (human) origin.

Kinetics of the reactions of purine nucleoside phosphorylases (PNP) from E. coli (PNP-I, the product of the deoD gene) and human erythrocytes with their natural substrates guanosine (Guo), inosine (Ino), a substrate analogue N(7)-methylguanosine (m(7)Guo), and orthophosphate (P(i), natural cosubstrate) and its thiophosphate analogue (SP(i)), found to be a weak cosubstrate, have been studied in the pH range 5-8. In this pH range Guo and Ino exist predominantly in the neutral forms (pK(a) 9.2 and 8.8); m(7)Guo consists of an equilibrium mixture of the cationic and zwitterionic forms (pK(a) 7.0); and P(i) and SP(i) exhibit equilibria between monoanionic and dianionic forms (pK(a) 6.7 and 5.4, respectively). The phosphorolysis of m(7)Guo (at saturated concentration) with both enzymes exhibits Michaelis kinetics with SP(i), independently of pH. With P(i), the human enzyme shows Michaelis kinetics only at pH approximately 5. However, in the pH range 5-8 for the bacterial enzyme, and 6-8 for the human enzyme, enzyme kinetics with P(i) are best described by a model with high- and low-affinity states of the enzymes, denoted as enzyme-substrate complexes with one or two active sites occupied by P(i), characterized by two sets of enzyme-substrate dissociation constants (apparent Michaelis constants, K (m1) and K (m2)) and apparent maximal velocities (V (max1) and V (max2)). Their values, obtained from non-linear least-squares fittings of the Adair equation, were typical for negative cooperativity of both substrate binding (K (m1) < K (m2)) and enzyme kinetics (V (max1)/K (m1) > V (max2)/K (m2)). Comparison of the pH-dependence of the substrate properties of P(i) versus SP(i) points to both monoanionic and dianionic forms of P(i) as substrates, with a marked preference for the dianionic species in the pH range 5-8, where the population of the P(i) dianion varies from 2 to 95%, reflected by enzyme efficiency three orders of magnitude higher at pH 8 than that at pH 5. This is accompanied by an increase in negative cooperativity, characterized by a decrease in the Hill coefficient from n (H) approximately 1 to n (H) approximately 0.7 for Guo with the human enzyme, and to n (H) approximately 0.7 and 0.5 for m(7)Guo with the E. coli and human enzymes, respectively. Possible mechanisms of cooperativity are proposed. Attention is drawn to the substrate properties of SP(i) in relation to its structure.

Contrasting effect of dark-chilling on chloroplast structure and arrangement of chlorophyll-protein complexes in pea and tomato: plants with a different susceptibility to non-freezing temperature.

The effect of dark-chilling and subsequent photoactivation on chloroplast structure and arrangements of chlorophyll-protein complexes in thylakoid membranes was studied in chilling-tolerant (CT) pea and in chilling-sensitive (CS) tomato. Dark-chilling did not influence chlorophyll content and Chl a/b ratio in thylakoids of both species. A decline of Chl a fluorescence intensity and an increase of the ratio of fluorescence intensities of PSI and PSII at 120 K was observed after dark-chilling in thylakoids isolated from tomato, but not from pea leaves. Chilling of pea leaves induced an increase of the relative contribution of LHCII and PSII fluorescence. A substantial decrease of the LHCII/PSII fluorescence accompanied by an increase of that from LHCI/PSI was observed in thylakoids from chilled tomato leaves; both were attenuated by photoactivation. Chlorophyll fluorescence of bright grana discs in chloroplasts from dark-chilled leaves, detected by confocal laser scanning microscopy, was more condensed in pea but significantly dispersed in tomato, compared with control samples. The chloroplast images from transmission-electron microscopy revealed that dark-chilling induced an increase of the degree of grana stacking only in pea chloroplasts. Analyses of O-J-D-I-P fluorescence induction curves in leaves of CS tomato before and after recovery from chilling indicate changes in electron transport rates at acceptor- and donor side of PS II and an increase in antenna size. In CT pea leaves these effects were absent, except for a small but irreversible effect on PSII activity and antenna size. Thus, the differences in chloroplast structure between CS and CT plants, induced by dark-chilling are a consequence of different thylakoid supercomplexes rearrangements.

Interpretation of fluorescence decay kinetics in 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides: exponential dependence on the number of phosphates in the polyphosphate bridge.

The number of phosphate groups in the 5',5'-polyphosphate bridge of mRNA-cap dinucleotide analogues affects kinetics of long-range electron transfer (ET) responsible for 3-methylbenzimidazole (m(3)B) fluorescence quenching in model dinucleotides. For instance, 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides (m(3)Bp( n )G, n = 2, 3, 4) having m(3)B donor, 5'-5' polyphosphate bridge, and guanine (G) acceptor, exhibit exponential dependence of the ET rate on the number of phosphates, i.e. donor-acceptor distance. Involvement of the 5'-5' polyphosphate bridge in the ET is strongly indicated by lack of m(3)B-G stacking effect on the exponential factor, which is the same at 20 degrees C, where m(3)B-G intramolecular stacking dominates, as that at 75 degrees C where stacking-unstacking equilibrium is shifted in favour of the unstacked structure.

A new approach to interpretation of heterogeneity of fluorescence decay: effect of induced tautomeric shift and enzyme-->ligand fluorescence resonance energy transfer.

Fluorescence decays in protein-ligand complexes are described by a new efficient model of continuous distribution of fluorescence lifetimes, and compared with multi-exponential models. Resulted analytical power-like decay function provides good fits to highly complex fluorescence kinetics. Moreover, this is a manifestation of so-called Tsallis q-exponential function, which is suitable for description of the systems with long-range interactions, memory effect, as well as with fluctuations of the characteristic lifetime of fluorescence. The proposed decay function was used to study effect of the interaction of E. coli purine nucleoside phosphorylase (PNP-I, the product of the deoD gene) with its specific inhibitor, viz. formycin A (FA), on fluorescence decays of ligand and enzyme tyrosine residues, in the presence of orthophosphate (P(i), a natural co-substrate). The power-like function provides new information about enzyme-ligand complex formation based on the excited state mean lifetime, heterogeneity parameter (q) and a number (N) of decay channels obtained from the variance of gamma distribution of fluorescence decay rates. With FA, which exists as a 85:15 mixture of the N(1)-H and N(2)-H tautomeric forms in aqueous solution, fluorescence intensity decay (lambda(exc)/lambda(em) 270/335 nm) is described by q approximately 1 and N approximately 200. Consequently power-like decay function converges to the single-exponential form, and lifetime distribution to the Dirac delta function. In contrast, selective excitation of the N(2)-H tautomer at higher wavelength led to a highly heterogenic fluorescence decay characterized by q>1 and 10-fold lower number of decay channels. Heterogeneity of fluorescence decays of both PNP-I and FA is enhanced by PNP-FA-P(i) complex formation, reflecting a shift of the tautomeric equilibrium of FA in favor of the N(2)-H species, and fluorescence resonance energy transfer (FRET) from protein tyrosine residue (Tyr160) to the bound N(2)-H tautomer. Moreover, proposed model is simple, and objectively describes heterogeneous nature of studied systems.

Photoisomerizations of N4-hydroxycytosines.

A series of N4-hydroxycytosines, unsubstituted or substituted with methyl groups at N3 or C5 atoms of the heterocyclic ring, was studied using the matrix-isolation method. Depending on the absence or presence of the methyl substituent at N3 or C5 atoms (or at both of them) the syn or anti form of the compounds (or a mixture of both forms) was trapped from the gas phase into a low-temperature matrix. Upon UV (lambda > 295 nm) irradiation of the matrixes the syn --> anti as well as the anti --> syn photoisomerization reactions were observed. The syn and anti isomers of N4-hydroxycytosines were identified by comparing their experimental IR spectra with the theoretical spectra calculated at the DFT(B3LYP)/6-31G(d,p) level. For the majority of the studied compounds, the UV induced reactions led to a photostationary state. The position of the final photostationary state was found to be a sensitive function of weak interactions of a studied N4-hydroxycytosine with the matrix environment: solid argon or solid nitrogen. However, not all of the studied photoisomerizations led to a classical photostationary state. For some of the investigated N4-hydroxycytosines, the position of the photostationary state was shifted very strongly in favor of the photoproduct, whereas for some others the position was shifted so strongly in favor of the starting isomer that no photoisomerization was observed. These experimental findings were elucidated by theoretical investigations of the potential energy surfaces of the ground (S0) and first excited (S1) electronic states of N4-hydroxycytosine. The crucial result of these calculations (carried out at the CASSCF level) was the localization of a conical intersection between S0 and S1 at a structure with perpendicular orientation of the hydroxylimino group with respect to the heterocyclic ring.

Interpretation of intramolecular stacking effect on the fluorescence intensity decay of 3-methylbenzimidazolyl(5'-5')guanosine dinucleotides using a model of lifetime distribution.

Time-resolved fluorescence of 3-methylbenzimidazole (m(3)B) was used to study stacking interaction between base moieties in di-, tri- and tetra-phosphate analogues of 3-methylbenzimidazolyl(5'-5')guanosine (m(3)Bp( n )G, n = 2, 3, 4), using 5'-triphosphate of 3-methylbenzimidazole riboside (m(3)BTP) as reference. Fluorescence intensity decays of all compounds cannot be satisfactory fitted with single-exponential function. Although an increase of a number of exponents led to better fits, interpretation of the individual exponential terms, i.e. pre-exponential amplitudes and fluorescence lifetimes, cannot be adequately characterized. We show that these fluorescence decays are best fitted by power-like function derived from physically justified distribution of the fluorescence lifetimes, and characterized by the mean value of the excited-state lifetime and relative variance of lifetime fluctuations around the mean value. The latter led to the parameter of heterogeneity and number of decay paths, which depend on the factors responsible for non-radiative decay of the excited state, including base-base stacking interaction. This was studied by means of changes of temperature and the number of phosphate groups in dinucleotides. It was shown that the strongest effect of stacking interactions, characterized by lowest values of both fluorescence mean decay time and relative variance, occurs in the case of m(3)Bp(3)G containing the same number of phosphates as natural mRNA cap. The possible importance of these results for interpretation of the mechanism of function of the mRNA cap structure is discussed.

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes.

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 degrees C and subsequent 3-h photoactivation under white light (200 mumol photons m(-2) s(-1)) at 22 degrees C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F(v)/F(m)) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.

Escherichia coli purine nucleoside phosphorylase II, the product of the xapA gene.

Purine nucleoside phosphorylases (PNPs, E. C. 2.4.2.1) use orthophosphate to cleave the N-glycosidic bond of beta-(deoxy)ribonucleosides to yield alpha-(deoxy)ribose 1-phosphate and the free purine base. Escherichia coli PNP-II, the product of the xapA gene, is similar to trimeric PNPs in sequence, but has been reported to migrate as a hexamer and to accept xanthosine with comparable efficiency to guanosine and inosine, the usual physiological substrates for trimeric PNPs. Here, we present a detailed biochemical characterization and the crystal structure of E.coli PNP-II. In three different crystal forms, PNP-II trimers dimerize, leading to a subunit arrangement that is qualitatively different from the "trimer of dimers" arrangement of conventional high molecular mass PNPs. Crystal structures are compatible with similar binding modes for guanine and xanthine, with a preference for the neutral over the monoanionic form of xanthine. A single amino acid exchange, tyrosine 191 to leucine, is sufficient to convert E.coli PNP-II into an enzyme with the specificity of conventional trimeric PNPs, but the reciprocal mutation in human PNP, valine 195 to tyrosine, does not elicit xanthosine phosphorylase activity in the human enzyme.

Xanthine, xanthosine and its nucleotides: solution structures of neutral and ionic forms, and relevance to substrate properties in various enzyme systems and metabolic pathways.

The 6-oxopurine xanthine (Xan, neutral form 2,6-diketopurine) differs from the corresponding 6-oxopurines guanine (Gua) and hypoxanthine (Hyp) in that, at physiological pH, it consists of a approximately 1:1 equilibrium mixture of the neutral and monoanionic forms, the latter due to ionization of N(3)-H, in striking contrast to dissociation of the N(1)-H in both Gua and Hyp at higher pH. In xanthosine (Xao) and its nucleotides the xanthine ring is predominantly, or exclusively, a similar monoanion at physiological pH. The foregoing has, somewhat surprisingly, been widely overlooked in studies on the properties of these compounds in various enzyme systems and metabolic pathways, including, amongst others, xanthine oxidase, purine phosphoribosyltransferases, IMP dehydrogenases, purine nucleoside phosphorylases, nucleoside hydrolases, the enzymes involved in the biosynthesis of caffeine, the development of xanthine nucleotide-directed G proteins, the pharmacological properties of alkylxanthines. We here review the acid/base properties of xanthine, its nucleosides and nucleotides, their N-alkyl derivatives and other analogues, and their relevance to studies on the foregoing. Included also is a survey of the pH-dependent helical forms of polyxanthylic acid, poly(X), its ability to form helical complexes with a broad range of other synthetic homopolynucleotides, the base pairing properties of xanthine in synthetic oligonucleotides, and in damaged DNA, as well as enzymes involved in circumventing the existence of xanthine in natural DNA.

Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence.

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P(i), substrate). Formation of enzyme-FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P(i). The effects of enzyme-FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)-H tautomer (predominant in solution) to the N(2)-H form, enhanced by the presence of P(i). The latter was confirmed by enzyme-ligand dissociation constant ( K(d)) values of 5.9+/-0.4 and 2.1+/-0.3 microM in the absence and presence of P(i), respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity ( K(d) approximately 70 microM), without changes in binding stoichiometry. Enzyme-FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)-H tautomer is characterized by a weaker phosphorescence than the N(1)-H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.