Room temperature luminescence of 1,N2-etheno-2-aminopurine in poly (vinyl alcohol) films.

We studied spectral properties of 1,N2-etheno-2-aminopurine after immobilization in poly (vinyl alcohol) films. The absorption spectrum of 1,N2-ε2APu consists of two peaks centered at 300 and 370 nm, and the fluorescence spectrum has maximum at about 460 nm. The fluorescence quantum efficiency is 62%. The fluorescence anisotropy reaches a value of 0.3 at longer wavelengths, while it is low at shorter wavelengths (corresponding to the second single excited state). The 1,N2-ε2APu has a relatively long fluorescence lifetime of about 16 ns and a noticeable room temperature phosphorescence with a lifetime of about 220 ms. A broad phosphorescence emission band (425-675 nm) is centered at about 530 nm and markedly overlaps with fluorescence at shorter wavelengths. Surprisingly, the phosphorescence excitation spectrum of 1,N2-ε2APu-doped poly (vinyl alcohol) film differs from the absorption and fluorescence excitation spectra. The strongest room temperature phosphorescence excitation is about 335 nm. At longer excitation wavelengths, above 450 nm, where fluorescence cannot be excited, a triplet excitation is still possible. The 1,N2-ε2APu phosphorescence anisotropy spectra confirm direct triplet state excitation. The ability to excite molecules at long wavelengths can find applications in the study of biological molecules that are unstable when excited at high energies.

Location Is Everything: Influence of His-Tag Fusion Site on Properties of Adenylosuccinate Synthetase from Helicobacter pylori.

The requirement for fast and dependable protein purification methods is constant, either for functional studies of natural proteins or for the production of biotechnological protein products. The original procedure has to be formulated for each individual protein, and this demanding task was significantly simplified by the introduction of affinity tags. Helicobacter pylori adenylosuccinate synthetase (AdSS) is present in solution in a dynamic equilibrium of monomers and biologically active homodimers. The addition of the His6-tag on the C-terminus (C-His-AdSS) was proven to have a negligible effect on the characteristics of this enzyme. This paper shows that the same enzyme with the His6-tag fused on its N-terminus (N-His-AdSS) has a high tendency to precipitate. Circular dichroism and X-ray diffraction studies do not detect any structural change that could explain this propensity. However, the dynamic light scattering, differential scanning fluorimetry, and analytical ultracentrifugation measurements indicate that the monomer of this construct is prone to aggregation, which shifts the equilibrium towards the insoluble precipitant. In agreement, enzyme kinetics measurements showed reduced enzyme activity, but preserved affinity for the substrates, in comparison with the wild-type and C-His-AdSS. The presented results reinforce the notion that testing the influence of the tag on protein properties should not be overlooked.

Trimeric Architecture Ensures the Stability and Biological Activity of the Calf Purine Nucleoside Phosphorylase: In Silico and In Vitro Studies of Monomeric and Trimeric Forms of the Enzyme.

Mammalian purine nucleoside phosphorylase (PNP) is biologically active as a homotrimer, in which each monomer catalyzes a reaction independently of the others. To answer the question of why the native PNP forms a trimeric structure, we constructed, in silico and in vitro, the monomeric form of the enzyme. Molecular dynamics simulations showed different geometries of the active site in the non-mutated trimeric and monomeric PNP forms, which suggested that the active site in the isolated monomer could be non-functional. To confirm this hypothesis, six amino acids located at the interface of the subunits were selected and mutated to alanines to disrupt the trimer and obtain a monomer (6Ala PNP). The effects of these mutations on the enzyme structure, stability, conformational dynamics, and activity were examined. The solution experiments confirmed that the 6Ala PNP mutant occurs mainly as a monomer, with a secondary structure almost identical to the wild type, WT PNP, and importantly, it shows no enzymatic activity. Simulations confirmed that, although the secondary structure of the 6Ala monomer is similar to the WT PNP, the positions of the amino acids building the 6Ala PNP active site significantly differ. These data suggest that a trimeric structure is necessary to stabilize the geometry of the active site of this enzyme.

Chromophore of an Enhanced Green Fluorescent Protein Can Play a Photoprotective Role Due to Photobleaching.

Under stress conditions, elevated levels of cellular reactive oxygen species (ROS) may impair crucial cellular structures. To counteract the resulting oxidative damage, living cells are equipped with several defense mechanisms, including photoprotective functions of specific proteins. Here, we discuss the plausible ROS scavenging mechanisms by the enhanced green fluorescent protein, EGFP. To check if this protein could fulfill a photoprotective function, we employed electron spin resonance (ESR) in combination with spin-trapping. Two organic photosensitizers, rose bengal and methylene blue, as well as an inorganic photocatalyst, nano-TiO2, were used to photogenerate ROS. Spin-traps, TMP-OH and DMPO, and a nitroxide radical, TEMPOL, served as molecular targets for ROS. Our results show that EGFP quenches various forms of ROS, including superoxide radicals and singlet oxygen. Compared to the three proteins PNP, papain, and BSA, EGFP revealed high ROS quenching ability, which suggests its photoprotective role in living systems. Damage to the EGFP chromophore was also observed under strong photo-oxidative conditions. This study contributes to the discussion on the protective function of fluorescent proteins homologous to the green fluorescent protein (GFP). It also draws attention to the possible interactions of GFP-like proteins with ROS in systems where such proteins are used as biological markers.

Tricyclic Nucleobase Analogs and Their Ribosides as Substrates and Inhibitors of Purine-Nucleoside Phosphorylases III. Aminopurine Derivatives.

Etheno-derivatives of 2-aminopurine, 2-aminopurine riboside, and 7-deazaadenosine (tubercidine) were prepared and purified using standard methods. 2-Aminopurine reacted with aqueous chloroacetaldehyde to give two products, both exhibiting substrate activity towards bacterial (E. coli) purine-nucleoside phosphorylase (PNP) in the reverse (synthetic) pathway. The major product of the chemical synthesis, identified as 1,N2-etheno-2-aminopurine, reacted slowly, while the second, minor, but highly fluorescent product, reacted rapidly. NMR analysis allowed identification of the minor product as N2,3-etheno-2-aminopurine, and its ribosylation product as N2,3-etheno-2-aminopurine-N2--D-riboside. Ribosylation of 1,N2-etheno-2-aminopurine led to analogous N2--d-riboside of this base. Both enzymatically produced ribosides were readily phosphorolysed by bacterial PNP to the respective bases. The reaction of 2-aminopurine-N9- -D-riboside with chloroacetaldehyde gave one major product, clearly distinct from that obtained from the enzymatic synthesis, which was not a substrate for PNP. A tri-cyclic 7-deazaadenosine (tubercidine) derivative was prepared in an analogous way and shown to be an effective inhibitor of the E. coli, but not of the mammalian enzyme. Fluorescent complexes of amino-purine analogs with E. coli PNP were observed.

Tri-Cyclic Nucleobase Analogs and their Ribosides as Substrates of Purine-Nucleoside Phosphorylases. II Guanine and Isoguanine Derivatives.

Etheno-derivatives of guanine, O6-methylguanine, and isoguanine were prepared and purified using standard methods. The title compounds were examined as potential substrates of purine-nucleoside phosphorylases from various sources in the reverse (synthetic) pathway. It was found that 1,N2-etheno-guanine and 1,N6-etheno-isoguanine are excellent substrates for purine-nucleoside phosphorylase (PNP) from E. coli, while O6-methyl-N2,3-etheno-guanine exhibited moderate activity vs. this enzyme. The latter two compounds displayed intense fluorescence in neutral aqueous medium, and so did the corresponding ribosylation products. By contrast, PNP from calf spleens exhibited only modest activity towards 1,N6-etheno-isoguanine; the remaining compounds were not ribosylated by this enzyme. The enzymatic ribosylation of 1,N6-etheno-isoguanine using two forms of calf PNP (wild type and N243D) and E. coli PNP (wild type and D204N) gave three different products, which were identified on the basis of NMR analysis and comparison with the product of the isoguanosine reaction with chloroacetic aldehyde, which gave an essentially single compound, identified unequivocally as N9-riboside. With the wild-type E. coli enzyme as a catalyst, N9--d- and N7--d-ribosides are obtained in proportion ~1:3, while calf PNP produced another riboside, tentatively identified as N6--d-riboside. The potential application of various forms of PNP for synthesis of the tri-cyclic nucleoside analogs is discussed.

Part-of-the-sites binding and reactivity in the homooligomeric enzymes - facts and artifacts.

For a number of enzymes composed of several subunits with the same amino acid sequence, it was documented, or suggested, that binding of a ligand, or catalysis, is carried out by a single subunit. This phenomenon may be the result of a pre-existent asymmetry of subunits or a limiting case of the negative cooperativity, and is sometimes called "half-of-the-sites binding (or reactivity)" for dimers and could be called "part-of-the-sites binding (or reactivity)" for higher oligomers. In this article, we discuss molecular mechanisms that may result in "part-of-the-sites binding (and reactivity)", offer possible explanations why it may have a beneficial role in enzyme function, and point to experimental problems in documenting this behaviour. We describe some cases, for which such a mechanism was first reported and later disproved. We also give several examples of enzymes, for which this mechanism seems to be well documented, and profitable. A majority of enzymes identified in this study as half-of-the-sites binding (or reactive) use it in the flip-flop version, in which "half-of-the-sites" refers to a particular moment in time. In general, the various variants of the mechanism seems to be employed often by oligomeric enzymes for allosteric regulation to enhance the efficiency of enzymatic reactions in many key metabolic pathways.

In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori.

Adenylosuccinate synthetase (AdSS) is an enzyme at regulatory point of purine metabolism. In pathogenic organisms which utilise only the purine salvage pathway, AdSS asserts itself as a promising drug target. One of these organisms is Helicobacter pylori, a wide-spread human pathogen involved in the development of many diseases. The rate of H. pylori antibiotic resistance is on the increase, making the quest for new drugs against this pathogen more important than ever. In this context, we describe here the properties of H. pylori AdSS. This enzyme exists in a dimeric active form independently of the presence of its ligands. Its narrow stability range and pH-neutral optimal working conditions reflect the bacterium's high level of adaptation to its living environment. Efficient inhibition of H. pylori AdSS with hadacidin and adenylosuccinate gives hope of finding novel drugs that aim at eradicating this dangerous pathogen.

Site-Selective Ribosylation of Fluorescent Nucleobase Analogs Using Purine-Nucleoside Phosphorylase as a Catalyst: Effects of Point Mutations.

Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-ß-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-ß-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.

Purine nucleoside phosphorylase activity decline is linked to the decay of the trimeric form of the enzyme.

Homotrimeric mammalian purine nucleoside phosphorylase (PNP) plays a key role in the nucleoside and nucleotide metabolic salvage pathway. Each monomer in the active PNP trimer is composed of a central ß-sheet flanked by several α-helices. We investigated the stability of calf PNP using analytical ultracentrifugation, differential scanning calorimetry, circular dichroism, and UV absorption spectroscopy. The results demonstrate that the activity decline (due to protein aging after isolation from cells) of wild type PNP and its two mutants with point mutations in the region of monomer-monomer interface, is accompanied by a decrease of the population of the trimeric enzyme and an increase of the population of its aggregated forms. The data do not indicate a significant population of either folded or unfolded PNP monomers. The enzyme with specific activity lower than the maximal shows a decrease of the helical structure, which can make it prone to aggregation. The presence of phosphate stabilizes the enzyme but leads to a more pronounced aggregation above the melting temperature. These results suggest that the biological role of packing of the PNP monomers into a trimeric structure is to provide the stability of the enzyme since the monomers are not stable in solution.

Two fluorogenic substrates for purine nucleoside phosphorylase, selective for mammalian and bacterial forms of the enzyme.

Two nontypical nucleosides, 7-ß-D-ribosyl-2,6-diamino-8-azapurine and 8-ß-D-ribosyl-2,6-diamino-8-azapurine, have been found to exhibit moderately good, and selective, substrate properties toward calf and bacterial (Escherichia coli) forms of purine nucleoside phosphorylase (PNP). The former compound is effectively phosphorolysed by calf PNP and the latter by PNP from E. coli. Both compounds are fluorescent with λ(max) ∼ 425 to 430 nm, but the reaction product, 2,6-diamino-8-azapurine, emits in a different spectral region (λ(max) ∼ 363 nm) with nearly 40% yield, providing a strong fluorogenic effect at 350 to 360 nm.

Enzymatic synthesis of highly fluorescent 8-azapurine ribosides using a purine nucleoside phosphorylase reverse reaction: variable ribosylation sites.

Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-ß-d-riboside (λmax 365 nm), while for N8-ß-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.

The pursuit of new alternative ways to eradicate Helicobacter pylori continues: Detailed characterization of interactions in the adenylosuccinate synthetase active site.

Purine nucleotide synthesis is realised only through the salvage pathway in pathogenic bacterium Helicobacter pylori. Therefore, the enzymes of this pathway, among them also the adenylosuccinate synthetase (AdSS), present potential new drug targets. This paper describes characterization of His6-tagged AdSS from H. pylori. Thorough analysis of 3D-structures of fully ligated AdSS (in a complex with guanosine diphosphate, 6-phosphoryl-inosine monophosphate, hadacidin and Mg2+) and AdSS in a complex with inosine monophosphate (IMP) only, enabled identification of active site interactions crucial for ligand binding and enzyme activity. Combination of experimental and molecular dynamics (MD) simulations data, particularly emphasized the importance of hydrogen bond Arg135-IMP for enzyme dimerization and active site formation. The synergistic effect of substrates (IMP and guanosine triphosphate) binding was suggested by MD simulations. Several flexible elements of the structure (loops) are stabilized by the presence of IMP alone, however loops comprising residues 287-293 and 40-44 occupy different positions in two solved H. pylori AdSS structures. MD simulations discovered the hydrogen bond network that stabilizes the closed conformation of the residues 40-50 loop, only in the presence of IMP. Presented findings provide a solid basis for the design of new AdSS inhibitors as potential drugs against H. pylori.

Trimeric purine nucleoside phosphorylase: exploring postulated one-third-of-the-sites binding in the transition state.

Transition-state analogue inhibitors, immucillins, were reported to bind to trimeric purine nucleoside phosphorylase (PNP) with the stoichiometry of one molecule per enzyme trimer [Miles, R. W.; Tyler, P. C.; Furneaux, R. H.; Bagdassarian, C. K.; Schramm, V. L. Biochem. 1998, 37, 8615]. In attempts to observe and better understand the nature of this phenomenon we have conducted calorimetric titrations of the recombinant calf PNP complexed with immucillin H. However, by striking contrast to the earlier reports, we have not observed negative cooperativity and we got the stoichiometry of three immucillin molecules per enzyme trimer. Similar results were obtained from fluorimetric titrations, and for other inhibitors bearing features of the transition state. However, we observed apparent cooperativity between enzyme subunits and apparent lower stoichiometry when we used the recombinant enzyme not fully purified from hypoxanthine, which is moped from Escherichia coli cells. Results presented here prove that one-third-of-the-sites binding does not occur for trimeric PNP, and give the highly probable explanation why previous experiments were interpreted in terms of this phenomenon.

Searching for Hydrodynamic Orienting Effects in the Association of Tri-N-acetylglucosamine with Hen Egg-White Lysozyme.

Using stopped-flow fluorometry, we determined rate constants for the formation of diffusional encounter complexes of tri-N-acetylglucosamine (NAG3) with hen egg-white lysozyme (kaWT) and its double mutant Asp48Asn/Lys116Gln (kaMT). We defined binding anisotropy, κ ≡ (kaWT - kaMT)/(kaWT + kaMT), and determined its ionic strength dependence. Our goal was to check if this ionic strength dependence provides information about the orienting hydrodynamic effects in the ligand-binding process. We also computed ionic strength dependence of the binding anisotropy from Brownian dynamics simulations using simple models of the lysozyme-NAG3 system. The results of our experiments indicate that in the case of lysozyme and NAG3 such hydrodynamic orienting effects are rather negligible. On the other hand, the results of our Brownian dynamics simulations prove that there exist molecular systems for which such orienting effects are substantial. However, the ionic strength dependence of the rate constants for the wild-type and modified systems do not exhibit any qualitative features that would allow us to conclude the presence of hydrodynamic orienting effects from stopped-flow experiments alone. Nevertheless, the results of our simulations suggest the presence of hydrodynamic orienting effects in the receptor-ligand association when the anisotropy of binding depends on the solvent viscosity.

Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent.

E. coli purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein-ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.

Overexpressed proteins may act as mops removing their ligands from the host cells: a case study of calf PNP.

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes--moping of ligands from the host cells--may take place in the case of other proteins with high overexpression yield.

1.45 A resolution crystal structure of recombinant PNP in complex with a pM multisubstrate analogue inhibitor bearing one feature of the postulated transition state.

Low molecular mass purine nucleoside phosphorylases (PNPs, E.C. 2.4.2.1) are homotrimeric enzymes that are tightly inhibited by immucillins. Due to the positive charge on the ribose like part (iminoribitol moiety) and protonation of the N7 atom of the purine ring, immucillins are believed to act as transition state analogues. Over a wide range of concentrations, immucillins bind with strong negative cooperativity to PNPs, so that only every third binding site of the enzyme is occupied (third-of-the-sites binding). 9-(5',5'-difluoro-5'-phosphonopentyl)-9-deazaguanine (DFPP-DG) shares with immucillins the protonation of the N7, but not the positive charge on the ribose like part of the molecule. We have previously shown that DFPP-DG interacts with PNPs with subnanomolar inhibition constant. Here, we report additional biochemical experiments to demonstrate that the inhibitor can be bound with the same K(d) ( approximately 190pM) to all three substrate binding sites of the trimeric PNP, and a crystal structure of PNP in complex with DFPP-DG at 1.45A resolution, the highest resolution published for PNPs so far. The crystals contain the full PNP homotrimer in the asymmetric unit. DFPP-DG molecules are bound in superimposable manner and with full occupancies to all three PNP subunits. Thus the postulated third-of-the-sites binding of immucillins should be rather attribute to the second feature of the transition state, ribooxocarbenium ion character of the ligand or to the coexistence of both features characteristic for the transition state. The DFPP-DG/PNP complex structure confirms the earlier observations, that the loop from Pro57 to Gly66 covering the phosphate-binding site cannot be stabilized by phosphonate analogues. The loop from Glu250 to Gln266 covering the base-binding site is organized by the interactions of Asn243 with the Hoogsteen edge of the purine base of analogues bearing one feature of the postulated transition state (protonated N7 position).

Analytical ultracentrifugation as a tool in the studies of aggregation of the fluorescent marker, Enhanced Green Fluorescent Protein.

Enhanced green fluorescent protein (EGFP) is a fluorescent marker used in bio-imaging applications, including as an indicator of folding or aggregation of a fused partner. However, the limited maturation, low folding efficiency, and presence of non-fluorescent states of EGFP can influence the interpretation of experimental data. To measure aggregation associated with de novo folding of EGFP from a high GdnHCl concentration, the analytical ultracentrifugation method was used. Absorption detection at 280 nm allowed to monitor the presence of monomers and aggregated forms. Fluorescence detection enabled the observation of only properly folded molecules with a functional chromophore. The results showed intensive aggregation of EGFP in low concentrations of GdnHCl with a continuous distribution of aggregated forms. The properly folded monomers with mature chromophore were fluorescent, while the conglomerates of EGFP molecules were not. These facts are essential for a proper interpretation of data obtained with EGFP labelling.

Heterodimerizing helices as tools for nanoscale control of the organization of protein-protein and protein-quantum dots.

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the Kds measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots.