NMR analysis of duplex d(CGCGATCGCG)<Subscript>2</Subscript> modified by <Emphasis Type="Italic">Λ</Emphasis>- and <Emphasis Type="Italic">Δ</Emphasis>-[Ru(bpy)<Subscript>2</Subscript>(m-GHK)]Cl<Subscript>2</Subscript> and DNA photocleavage study

The interaction of the diastereomeric complexes Λ-[Ru(bpy)₂(m-GHK)]Cl₂ and Δ-[Ru(bpy)₂(m-GHK)]Cl₂ (bpy is 2,2′-bipyridine, GHK is glycine–l-histidine–l-lysine) with the deoxynucleotide duplex d(5′-CGCGATCGCG)₂ was studied by means of ¹H NMR spectroscopy. At a Δ-isomer to DNA ratio of 1:1, significant shifts for the metal complex are observed, whereas there is negligible effect on the oligonucleotide protons and only one intermolecular nuclear Overhauser effect (NOE) is present at the 2D nuclear Overhauser enhancement spectroscopy spectrum. The ¹Η NMR spectrum at ratio 2:1 is characterized by a slight shift for the Δ-isomer’s bpy aromatic protons as well as significant shifts for the decanucleotide G₄ H1′ and Η2″, A₅ H2, G₁₀ H1′, T₆ NH and G₂ NH protons. Furthermore, at ratio 2:1, 11 intermolecular NOEs are observed. The majority of the NOEs involve the sugar Η2′ and Η2″ protons sited in the major groove of the decanucleotide. Increasing the Δ-isomer to d(CGCGATCGCG)₂ ratio to 5:1 results in noteworthy spectral changes. The Δ-isomer’s proton shifts are reduced, whereas significant shifts are observed for the decanucleotide protons, especially the sugar protons, as well as for the exchangeable protons. Interaction is characterized by the presence of only one intermolecular NOE. Furthermore, there is significant broadening of the imino proton signals as the ratio of the Δ-isomer to DΝΑ increases, which is attributed to the opening of the two strands of the duplex. The Λ-isomer, on the other hand, approaches the minor groove of the oligonucleotide and interacts only weakly, possibly by electrostatic interactions. Photocleavage studies were also conducted with the plasmid pUC19 and a 158-bp restriction fragment, showing that both diastereomers cleave DNA with similar efficiency, attacking mainly the guanines of the sequence probably by generating active oxygen species. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. An erratum to this article can be found at     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Simultaneous α/β spin-state selection for <Superscript>13</Superscript>C and <Superscript>15</Superscript>N from a time-shared HSQC-IPAP experiment

Two novel HSQC-IPAP approaches are proposed to achieve α/β spin-state editing simultaneously for ¹³C and ¹⁵N in a single NMR experiment. The pulse schemes are based on a time-shared (TS) 2D ¹H,¹³C/¹H,¹⁵N-HSQC correlation experiment that combines concatenated echo elements for simultaneous J(CH) and J(NH) coupling constants evolution, TS evolution of ¹³C and ¹⁵N chemical shifts in the indirect dimension and heteronuclear α/β-spin-state selection by means of the IPAP principle. Heteronuclear α/β-editing for all CH _n (n = 1–3) and NH _n (1–2) multiplicities can be achieved in the detected F2 dimension of a single TS-HSQC-F2-IPAP experiment. On the other hand, an alternative TS-HSQC-F1-IPAP experiment is also proposed to achieve α/β-editing in the indirect F1 dimension. Experimental and simulated data is provided to evaluate these principles in terms of sensitivity and performance simultaneously on backbone and side-chain CH, CH₂, CH₃, NH, and NH₂ spin systems in uniformly ¹³C/¹⁵N-labeled proteins and in small natural-abundance peptides.     

The cadmium binding domains in the metallothionein isoform Cd7-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy

The metal–thiolate connectivity of recombinant Cd₇-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of ¹⁵N T ₁ and T ₂ relaxation times and steady state {¹H}–¹⁵N heteronuclear NOEs. The ¹¹³Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of ¹¹³Cd NMR signals. The complete assignment of cysteine Hα and Hβ proton resonances and the analysis of 2D ¹¹³Cd–¹¹³Cd COSY and ¹H–¹¹³Cd HMQC type spectra allowed us to identify a four metal–thiolate cluster (α-domain) and a three metal–thiolate cluster (β-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the α and β domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the α-domain of mussel Cd₇-MT10 is of the form M₄S₁₂ instead of M₄S₁₁, and has a novel topology. The β-domain has a metal–thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the β-domain displays a more disordered conformation than the α-domain. It is concluded that in mussel Cd₇-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal–thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.     

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An in situ study of photosynthetic oxygen exchange and electron transport rate in the marine macroalga Ulva lactuca (Chlorophyta)

Direct comparisons between photosynthetic O₂ evolution rate and electron transport rate (ETR) were made in situ over 24 h using the benthic macroalga Ulva lactuca (Chlorophyta), growing and measured at a depth of 1.8 m, where the midday irradiance rose to 400–600 μmol photons m⁻² s⁻¹. O₂ exchange was measured with a 5-chamber data-logging apparatus and ETR with a submersible pulse amplitude modulated (PAM) fluorometer (Diving-PAM). Steady-state quantum yield ((F_m′−F_t)/F_m′) decreased from 0.7 during the morning to 0.45 at midday, followed by some recovery in the late afternoon. At low to medium irradiances (0–300 μmol photons m⁻² s⁻¹), there was a significant correlation between O₂ evolution and ETR, but at higher irradiances, ETR continued to increase steadily, while O₂ evolution tended towards an asymptote. However at high irradiance levels (600–1200 μmol photons m⁻² s⁻¹) ETR was significantly lowered. Two methods of measuring ETR, based on either diel ambient light levels and fluorescence yields or rapid light curves, gave similar results at low to moderate irradiance levels. Nutrient enrichment (increases in [NO₃ ⁻], [NH₄ ⁺] and [HPO₄ ^2-] of 5- to 15-fold over ambient concentrations) resulted in an increase, within hours, in photosynthetic rates measured by both ETR and O₂ evolution techniques. At low irradiances, approximately 6.5 to 8.2 electrons passed through PS II during the evolution of one molecule of O₂, i.e., up to twice the theoretical minimum number of four. However, in nutrient-enriched treatments this ratio dropped to 5.1. The results indicate that PAM fluorescence can be used as a good indication of the photosynthetic rate only at low to medium irradiances. This revised version was published online in June 2006 with corrections to the Cover Date.     

Kinetics of formation and stability of {Pt(dien)}2+ complexes with octamer and 14-mer DNA oligonucleotides containing a GG sequence

 Reaction of [Pt(dien)Cl]⁺ (1) with the 14-mer oligonucleotide 5′-d(ATACATGGTACATA) (I) gave rise to two major species which corresponded to the 5′-G and 3′-G platinated monofunctional adducts, and a minor amount of the bis-platinated adduct formed during the later stages of the reaction. The reaction of (1) with the related octamer 5′-d(ATACATGG) (II) was also investigated. Kinetic data obtained by HPLC showed that the 5′-G and 3′-G bases of the 14-mer oligonucleotide were platinated at similar rates: the second-order rate constant is 53×10^–2 M^–1 s^–1 at 298 K in 0.1 M NaClO₄. However, the platination rate of 5′-G of the octamer (II) (k=69×10^–2 M^–1 s^–1) was enhanced by a factor of three compared to the rate of platination at 3′-G (k=22×10^–2 M^–1 s^–1). All the adducts were separated by HPLC and characterized by NMR spectroscopy, enzymatic digestion and MALDI-TOF mass spectrometry. ¹H and ¹⁵N NMR shifts suggest that there are distinct conformational differences between 14-mer duplexes platinated at 5′-G (I5′ _ds) and 3′–G (I3′ _ds). Molecular mechanics modelling indicates that rotation around the Pt-N7 bond is more restricted in the case of the 5′-G adduct than in that of the 3′-G adduct. The binding of {Pt(dien)}²⁺ to 5′-GN7 and 3′-GN7 in the monofunctional adducts of (I) was shown to be reversible upon the addition of high concentrations of chloride ions. Received: 3 July 1998 / Accepted: 10 November 1998     

Modulation of Bacillus pasteurii cytochrome c 553 reduction potential by structural and solution parameters

 Direct cyclic voltammetry and ¹H NMR spectroscopy have been combined to investigate the electrochemical and spectroscopic properties of cytochrome c ₅₅₃ isolated from the alkaliphilic soil bacterium Bacillus pasteurii. A quasi-reversible diffusion-controlled redox process is exhibited by cytochrome c ₅₅₃ at a pyrolitic graphite edge microelectrode. The temperature dependence of the reduction potential, measured using a non-isothermal electrochemical cell, revealed a discontinuity at 308 K. The thermodynamic parameters determined in the low-temperature range (275–308 K;ΔS°′=–162.7±1.2 J mol^–1 K^–1, ΔH°′=–53.0±0.5 kJ mol^–1, ΔG°′=–4.5±0.1 kJ mol^–1, E°′=+47.0±0.6 mV) indicate the presence of large enthalpic and entropic effects, leading, respectively, to stabilization and destabilization of the reduced form of cytochrome c ₅₅₃. Both effects are more accentuated in the high-temperature range (308–323 K;ΔS°′=–294.1±8.4 J mol^–1 K^–1, ΔH°′=–93.4±3.1 kJ mol^–1, ΔG°′=–5.8±0.6 kJ mol^–1, E°′=+60.3±5.8 mV), with the net result being a slight increase of the standard reduction potential. These thermodynamic parameters are interpreted using the compensation theory of hydration of biopolymers as indicating the extrusion, upon reduction, of water molecules from the hydration sphere of the cytochrome. The low-T and high-T conformers differ by the number of water molecules in the solvation sphere: in the high-T conformer, the number of water molecules extruded upon reduction increases, as compared to the low-T conformer. The ionic strength dependence of the reduction potential at 298 K, treated within the frame of extended Debye-Hückel theory, yields values of E ^°′ _(I=0) =–25.4±1.4 mV, z _red=–11.3, and z _ox=–10.3. The pH dependence of the reduction potential at 298 K shows a plateau in the pH range 7–10 and an increase at more acidic pH, allowing the calculation of pK _O=5.5 and pK _R=5.7, together with the estimate of the reduction potentials of completely protonated (+71 mV) and deprotonated (+58 mV) forms of cytochrome c ₅₅₃. ¹H NMR spectra of the oxidized paramagnetic cytochrome c ₅₅₃ indicate the presence of a His-Met axial coordination of the low-spin (S=1/2) heme iron, which is maintained in the temperature interval 288–340 K at pH 7 and in the pH range 4.8–10.0 at 298 K. The temperature dependence of the hyperfine-shifted signals shows both Curie-type and anti-Curie-type behavior, with marked deviations from linearity, interpreted as indicating the presence of a fast equilibrium between the low-T and high-T conformers, having slightly different heme electronic structures resulting from the T-induced conformational change. Increasing the NaCl concentration in the range 0–0.2 M causes a slight change of the ¹H NMR chemical shifts of the hyperfine-shifted signals, with no influence on their linewidth. The calculated lower limit value of the apparent affinity constant for specific ion binding is estimated as 5.2±1.1 M^–1. The pH dependence of the isotropically shifted ¹H NMR signals of the oxidized cytochrome displays at least one ionization step with pK _O=5.7. The thermodynamic and spectroscopic data indicate a large solvent-derived entropic effect as the main cause for the observed low reduction potential of B. pasteurii cytochrome c ₅₅₃. Received: 9 January 1998 / Accepted: 8 April 1998     

Multiple quadrature detection in reduced dimensionality experiments

A new, simple procedure is proposed which enables acquisition of two or more chemical shifts encoded in a common dimension simultaneously in quadrature. For n chemical shifts projected in a single dimension, the expected effect is obtained by interleaved acquisition and appropriate combination of 2ⁿ data sets per increment of respective evolution time. The particular chemical shifts can be calculated from sums and differences of signal frequencies obtained by different combination of the acquired data sets. In comparison to the established reduced dimensionality (RD) techniques, the proposed method enhances resolution due to reduction of the number of signals and requires less evolution time increments owing to narrower spectral width in the RD-domain. We show examples of the application of the new approach to the 2D HNCA and HN(CO)CA techniques with two, and 2D HACANH with three frequencies simultaneously encoded in the t ₁ evolution period, for ¹³C,¹⁵N-labeled ubiquitin.     

Modulation of the Calmodulin-induced Inhibition of Sarcoplasmic Reticulum Calcium Release Channel (Ryanodine Receptor) by Sulfhydryl Oxidation in Single Channel Current Recordings and [3H]Ryanodine Binding

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl–sulfonic acid (4-CMPS) and 4,4′-dithiodipyridine (4,4′-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [³H]ryanodine binding to HSR vesicles. 0.1 μm CaM reduced the open probability (P _o ) of the calcium release channel at maximally activating calcium concentrations (50–100 μm) from 0.502 ± 0.02 to 0.137 ± 0.022 (n= 28), with no effect on unitary conductance. 4-CMPS (10–40 μm) and 4,4′-DTDP (0.1–0.3 mm) induced a concentration dependent increase in P _o (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4′-DTDP to higher concentrations in single channel recordings and [³H]ryanodine binding. 40 μm 4-CMPS induced a near maximal (P _o > 0.9) and 0.3 mm 4,4′-DTDP a submaximal (P _o = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4′-DTDP affected Ca-[¹²⁵I]calmodulin binding to HSR. 1 mm MgCl₂ reduced P _o from 0.53 to 0.075 and 20–40 μm 4-CMPS induced a near maximal channel activation (P _o > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4′-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel. Received: 22 July 1999/Revised: 15 November 1999     

Cis-diammineplatinum(II) forms a macrochelate with 2′-deoxycytidine 5′-monophosphate (dCMP2–)! Reactivity and acid-base properties of cis-Pt(NH3)2(dCMP)

 The synthesis of cis-Pt(NH₃)₂(dCMP) is reported and by various physico-chemical methods it is demonstrated that it is a macrochelate in which Pt(II) is bound simultaneously to the N3 site of cytosine in dCMP^2– and to a phosphate-oxygen atom. According to the NOESY spectra (cross-peaks between cytosine H6 and H2′ and H3′) the cytosine ring adopts an anti orientation. Highly unusual is the significant (1 ppm) downfield shift of the sugar proton H5″ in the ¹H-NMR spectrum and the sensitivity of the cytosine H6 resonance on the protonation state of the phosphate group. Based on these three features a geometry for the macrochelate is proposed. The compound is a major product of the reaction of cis-[Pt(NH₃)₂(H₂O)₂]²⁺ with dCMP^2– at neutral pH, but it even forms at pH 5. By applying pD-dependent NMR spectroscopy (¹H, ³¹P) and potentiometric pH titration, it is demonstrated that the Pt-coordinated phosphate group can be protonated (pK _a/1=3.21±0.10 and 3.31±0.05, respectively), and ¹H- and ³¹P-NMR spectra also indicate deprotonation (pK _a/2=13.35±0.25) of the exocyclic amino group of the cytosine moiety. The metal ion binding affinity of cis-Pt(NH₃)₂(dCMP) is very small, as shown for Cu²⁺ (log K<0.6). The cis-Pt(NH₃)₂(dCMP) complex reacts with nucleosides and nucleotides (L′) by losing its chelate structure and forming mixed ligand complexes, cis-Pt(NH₃)₂(dCMP)(L′); this means that the phosphate group is released from the coordination sphere of Pt(II), indicating that the Pt(II)-O(phosphate) bond is not very strong. Received: 23 October 1997 / Accepted: 17 February 1998     

Catalysis and Selectivity in Prebiotic Synthesis: Initiation of the Formation of Oligo(U)s on Montmorillonite Clay by Adenosine-5′-methylphosphate

Adenosine-5′-methylphosphate (MepA) initiates the oligomerization of the 5′-phosphorimidazolide of uridine (ImpU) in the presence of montmorillonite clay. Longer oligomers form because the 5′-phosphate is blocked with a methyl group that prevents the formation of cyclic- and pyrophosphate-containing compounds. The MepA initiates 69–84% of the 5–9 charge oligomers, respectively. The montmorillonite catalyst also provides selectivity in the oligomerization reactions so that the main reaction pathway is MepA → MepA³′pU → MepA³′pU²′pU → MepA³′pU²′pU³′pU. MepA did not enhance the oligomerization of ImpA. The relative rates of the reactions were determined from an investigation of the products in competitive reactions. Selectivity was observed in the reaction of ImpU with equimolar amounts of MepA³′pU and MepA²′pU where the relative reaction rates are 10.3:1, respectively. In the reaction of ImpA with MepA³′pA and MepA²′pA the ImpA reacts 5.2 times faster with MepA³′pA. In the competitive reaction of ImpU and ImpA with MepA³′pA and MepA³′pU the elongation proceeds on MepA³′pA 5.6 times more rapidly than with MepA³′pU. There is no correlation between the extent of binding to the montmorillonite and reaction rates in the formation of longer oligomers. The formation of more than two sequential 2′,5′-linkages in the oligomer chain proceeds more slowly than the addition to a single 2′,5′-link or a 3′,5′-link and either chain termination or elongation by a 3′,5′-linage occurs. The central role that catalysis may have had in the prebiotic formation of biopolymers is discussed. Note added in proof: There are errors in the high resolution mass spectral data given in Section 4.2.1. The high resolution mass spectrum found for the cyclic dimer of UpUp (C-UpUp) was 657.02260. C₁₈H₂₁N₄O₁₆P₂Na₂ requires 657.02232. The high resolution mass spectrum found for the cyclic dimer of ApAp (C-ApAp) was 725.05850. C₂₀H₂₂N₁₀O₁₂P₂Na₃ requires 725.05839.     

Two modes of sulfite oxidation in the extremely thermophilic and acidophilic archaeon Acidianus ambivalens

Sulfite-oxidizing enzyme activities were analyzed in cell-free extracts of aerobically grown cells of Acidianus ambivalens, an extremely thermophilic and chemolithoautotrophic archaeon. In the membrane and cytoplasmic fractions, two distinct enzyme activities were found. In the membrane fraction, a sulfite:acceptor oxidoreductase activity was found [530 mU (mg protein)^–1; apparent K _m for sulfite, 3.6 mM]. In the cytoplasmic fraction the following enzyme activities were found and are indicative of an oxidative adenylylsulfate pathway: adenylylsulfate reductase [138 mU (mg protein)^–1], adenylylsulfate:phosphate adenyltransferase [“ADP sulfurylase”; 86 mU (mg protein)^–1], adenylate kinase [650 mU (mg protein)^–1], and rhodanese [thiosulfate sulfur transferase, 9.2 mU (mg protein)^–1]. In addition, 5′,5′′′-P¹,P⁴-di(adenosine-5′) tetraphosphate (Ap₄A) synthase and Ap₄A pyrophosphohydrolase activities were detected. Received: 17 August 1998 / Accepted: 29 April 1999     

Sequential backbone assignment of uniformly 13C-labeled RNAs by a two-dimensional P(CC)H-TOCSY triple resonance NMR experiment

Summary A new ¹H−¹³C−³¹P triple resonance experiment is described which allows unambigous sequential backbone assignment in ¹³C-labeled oligonucleotides via through-bond coherence transfer from ³¹P via ¹³C to ¹H. The approach employs INEPT to transfer coherence from ³¹P to ¹³C and homonuclear TOCSY to transfer the ¹³C coherence through the ribose ring, followed by ¹³C to ¹H J-cross-polarisation. The efficiencies of the various possible transfer pathways are discussed. The most efficient route involves transfer of ³¹P_i coherence via C4′_i and C4′_i-1, because of the relatively large J′_PC4 couplings involved. Via the homonuclear and heteronuclear mixing periods, the C4′_i and C4′_i-1 coherences are subsequently transferred to, amongst others, H1′_i and H1′_i-1, respectively, leading to a 2D ¹H−³¹P spectrum which allows a sequential assignment in the ³¹P−¹H1′ region of the spectrum, i.e. in the region where the proton resonances overlap least. The experiment is demonstrated on a ¹³C-labeled RNA hairpin with the sequence 5′(GGGC-CAAA-GCCU)3′.     

19F NMR study on the biological Baeyer-Villiger oxidation of acetophenones

The biological Baeyer–Villiger oxidation of acetophenones was studied by ¹⁹F nuclear magnetic resonance (NMR). The ¹⁹F NMR method was used to characterise the time-dependent conversion of various fluorinated acetophenones in either whole cells of Pseudomonas fluorescens ACB or in incubations with purified 4′-hydroxyacetophenone monooxygenase (HAPMO). Whole cells of P. fluorescens ACB converted 4′-fluoroacetophenone to 4-fluorophenol and 4′-fluoro-2′-hydroxyacetophenone to 4-fluorocatechol without the accumulation of 4′-fluorophenyl acetates. In contrast to 4-fluorophenol, 4-fluorocatechol was further degraded as evidenced by the formation of stoichiometric amounts of fluoride anion. Purified HAPMO catalysed the strictly NADPH-dependent conversion of fluorinated acetophenones to fluorophenyl acetates. Incubations with HAPMO at pH 6 and 8 showed that the enzymatic Baeyer–Villiger oxidation occurred faster at pH 8 but that the phenyl acetates produced were better stabilised at pH 6. Quantum mechanical characteristics explained why 4′-fluoro-2′-hydroxyphenyl acetate was more sensitive to base-catalysed hydrolysis than 4′-fluorophenyl acetate. All together, ¹⁹F NMR proved to be a valid method to evaluate the biological conversion of ring-substituted acetophenones to the corresponding phenyl acetates, which can serve as valuable synthons for further production of industrially relevant chemicals. Journal of Industrial Microbiology & Biotechnology (2001) 26, 35–42. Received 20 April 2000/ Accepted in revised form 16 September 2000     

Hoxd1 is Expressed by Oligodendroglial Cells and Binds to a Region of the Human Myelin Oligodendrocyte Glycoprotein Promoter in vitro

(1) Little information exists on the role of clustered Hox genes in oligodendrocyte (OG) development. This study examines the expression profile of Hoxd1 and identifies a potential downstream target in the OG lineage. (2) Immunocytochemical analysis of primary mixed glial cultures demonstrated Hoxd1 was expressed throughout OG development. (3) A human myelin protein gene, myelin oligodendrocyte glycoprotein (MOG), was identified as a putative downstream target of Hoxd1 through Genbank searches utilizing the Hoxd1 homeodomain consensus binding sequence. (4) The dissociation coefficient constant (K _D) and dissociation rate constant (k _d) of the Hoxd1–MOG complex, determined using electrophoretic mobility shift assays (EMSAs), were estimated to be 1.9 × 10⁻⁷ M and 1.3 × 10⁻³ s⁻¹, respectively. The DNA–Hoxd1 homeodomain complex had a half-life (t _1/2) of 15 min. (5) Mutational analysis of Hoxd1–MOG complexes revealed the binding affinity of M1 (with mutation from ⁻¹⁰⁵⁴5′-TAAT-3′⁻¹⁰⁵¹ to TACT within the consensus binding site) and M2 (with mutation from ^-10545′-TAATTG-3′^-1049 to TAATCC within the consensus binding site) probes to the MOG promoter was severely affected. Thus the TAATTG core of the binding sequence appears important for Hoxd1 specificity. (6) Analysis of the involvement of TAAT sites adjacent to the consensus sequence in Hoxd1 binding showed the binding affinity of the M3 probe was affected, but not as severely as the M1 and M2 probes. These in vitro results suggest the TTTAATTGTA sequence is involved in Hoxd1 binding to the MOG promoter but neighboring TAAT sites may also be needed. Thus, MOG may be a target of Hoxd1.     

Effect of oral creatine ingestion on parameters of the work rate-time relationship and time to exhaustion in high-intensity cycling

The relationship between work rate (W˙) and time to exhaustion (t) during intense exercise is commonly described by either a hyperbolic function (NLin), t=W ^′/(W˙−W˙ _cp), or by its linear equivalent (LinW) W _lim =W ^′+W˙ _cp(t). The parameter W˙<INF _</INF>cp (critical power) has been described as an inherent characteristic of the aerobic energy system, while W ′ has been shown to be a ralid estimate of anaerobic work capacity. Recent studies have demonstrated that oral supplementation of creatine monohydrate (CrH₂O) increases total muscle creatine stores, and have linked these increases to improved performances in intense intermittent exercise. This study was conducted to determine the effect of CrH₂O supplementation on estimates of W ′ and W˙<INF _</INF>cp derived from the NLin and LinW equations, and to determine the effect of CrH₂O on t in exhaustive constant power exercise of different intensities. Fifteen active but untrained university students completed three phases of testing on a cycle ergometer: (1) familiarization, three learning trials, (2) baseline determination of W ′ and W˙<INF _</INF>cp, four bouts performed at a W˙ selected to elicit fatigue in 90–600 s, and (3) experimental determination of W ′ and W˙ _cp, four bouts performed at the same W˙ as baseline, but performed after 5 days of ingesting either a placebo (4 × 6 g of glucose/day) or CrH₂O (4 × 5 g of CrH₂O and 1 g glucose/day). Testing was administered in a double-blind manner. Analyses of covariance revealed a significant effect for CrH₂O on both estimates of W ′ (NLin, P=0.04; LinW, P<0.01), but not on estimates of W˙ _cp (NLin, P=0.37; LinW; P=0.30). Within groups, t was significantly different for only CrH₂O at the two highest W˙s (P=0.04). It is concluded that oral ingestion of CrH₂O increases estimates of W ′ due to an improved t at the shorter, more intense exercise bouts. Accepted: 1 September 1997     

Coherent phenomena of charge separation in reaction centers of LL131H and LL131H/LM160H/FM197H mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0–4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935–1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20–500 cm⁻¹. The most intense band has the frequency of −30 cm⁻¹ in RCs of mutant LL131H and in native RCs and the frequency of ∼100 cm⁻¹ in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B_A⁻ which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the B_A molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Extended Flip-back Schemes for Sensitivity Enhancement in Multidimensional HSQC-type Out-and-back Experiments

In many NMR experiments, only polarisation of a limited sub-set of all protons is converted into observable coherence. As recently shown by the “longitudinal” TROSY implementation (Pervushin et al. (2002) J. Am. Chem. Soc., 124, 12898–12902) and SOFAST-HMQC (Schanda and Brutscher (2005) J. Am. Chem. Soc., 127, 8014–8015), recovery of unused polarisation can be used indirectly and unspecifically to cool the proton lattice and, thus, accelerate re-equilibration for the selected proton subset. Here we illustrate transfer of this principle to HSQC-based multi-dimensional out-and-back experiments that exploit only polarisation of ¹⁵N-bound protons. The presented modifications to the pulse sequences can be implemented broadly and easily, extending standard flip-back of water polarisation to a much larger pool of protons that may comprise all non−¹⁵N-bound protons. The underlying orthogonal separation of H^N polarisation (selected by the main transfer path) from unused H^u polarisation (flipped-back on the recovery path) is thereby achieved through positive or negative selection by J-coupling, or using band-selective pulses. In practice, H^u polarisation recovery degrades mostly through cumulative pulse imperfections and transverse relaxation; we present, however, strategies to substantially minimise such losses particularly during interim proton decoupling. Depending on the protein’s relaxation properties and the extended flip-back scheme employed, we recovered up to 60% H^u equilibrium polarisation. The concomitant cooling of the proton lattice afforded substantial gains of more than 40%, relative to the water-only flip-back version, in the fast pulsing regime with re-equilibration delays τ much shorter than optimal (τ_opt = 1.25 · T₁(H^N)). These would be typically employed if resolution requirements dominate the total measurement time. Contrarily, if sensitivity is limiting and optimal interscan delays τ_opt can be set (optimal pulsing regime), the best of the presented flip-back schemes may still afford up to ca. 10% absolute sensitivity enhancement.