Dynamics of the N-terminal alpha-helix unfolding in the photoreversion reaction of phytochrome A

Time-resolved circular dichroism spectroscopy in the far-UV spectral region was used to examine the intermediates of the phytochrome photoreversion reaction (Pfr --> Pr). Three intermediates, lumi-F (tau = 320 ns), meta-Fa (tau = 265 micros) and meta-Fb (tau = 5.5 ms), have been identified in a simple sequential kinetic photoreversion mechanism by absorption spectroscopy [Linschitz, H., Kasche, V., Butler, W. L., & Siegelman, H. W. (1966) J. Biol. Chem. 241, 3395-3403; Pratt, L. H., & Butler, W. L. (1968) Photochem. Photobiol. 8, 477-485; Burke, M., Pratt, D. C., & Moscowitz, A. (1972) Biochemistry 11, 4025-4031; Spruit, C. J. P., Kendrick, R. E., & Cooke, R. J. (1975) Planta (Berlin) 127, 121-132; Eilfeld, P., & Rüdiger, W. (1985) Z. Naturforsch. 40c, 109-114; Chen, E., Lapko, V. N., Lewis, J. W., Song, P.-S., & Kliger, D. S. (1996) Biochemistry 35, 843-850]. In order to correlate the unfolding of the N-terminal alpha-helical segment with one or more of the intermediate species, time-resolved methods were coupled with the structurally sensitive probe of CD in the far-UV spectral region. Analysis of the TRCD data associates the decrease in alpha-helical content that occurs upon formation of Pr with decay of the meta-Fa intermediate. This unfolding process occurs with a time constant of 310 +/- 125 micros, which is consistent with the 265-micros lifetime for meta-Fa.     

Enantioselective analysis of praziquantel in plasma samples

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.     

A Note on CH3OD Spectra, Ground-State Energies and FIR Laser Assignments

Literature data on the CO-stretching and the ground-state torsion-rotation spectra of the CH₃OD isotopic species of methanol have been exploited to extrapolate the ground-state energies to higher J through use of ground-state combination differences derived from the CO-stretching wavenumbers. In particular, the J-dependence has been extended and better defined for the difficult |K| = 0 to 2 E energy level sequences, which are strongly affected by molecular asymmetry. The pattern of the J-dependence is found to be qualitatively well reproduced by a simple 11 × 11 Hamiltonian model incorporating ΔK = ±1 and ±2 off-diagonal asymmetry matrix elements. The results suggest partial reassignment of certain CO-stretching subbands, and the K = 8 and 9 A subbands have been newly identified through clues from reported far-infrared laser (FIRL) emission. Transition combination loops support a number of FIRL line assignments previously proposed, but show that the FIRL system pumped by the 9R(4) CO₂ line must be reassigned.     

The impact of EU policies on energy use in and the evolution of the UK built environment

Energy use in buildings is influenced by a variety of factors in complex ways. Historically, in the UK the efficiency of energy use in buildings has not been a major consideration in their design. Now policy concern with climate change is changing this, because buildings have come to be perceived as the locus of energy use with the highest cost-effective energy savings potential. In the UK, the energy efficiency of the building stock is rather low. The paper focuses largely on energy use in the UK's existing building stock and the two main European Union Directives which affect it: the Energy Performance in Buildings Directive and the Energy Services Directive. The Directives are complex, and there are a number of supporting programmes set up by the European Commission to aid their implementation. Even so, they have been implemented in differing ways in different European countries, and implementation remains patchy. The Directives have the potential to be a major influence on the evolution of the UK's built environment, but their effect will depend on the details of the Directives’ implementation and enforcement, many of which are not yet clear.     

The use of 18O as a tracer to study the growth mechanisms of oxide scales

A nuclear technique for the determination of ¹⁸O concentration as a function of depth is described. Examples are given of the application of this technique to studies of growth mechanisms of oxide scales.     

Formation of acetaldehyde from threonine by lactic acid bacteria.

Group N streptococci were found to cleave threonine to form acetaldehyde and glycine. Threonine aldolase, the enzyme catalysing this reaction, was found in all strains except Streptococcus cremoris Z8, an organism which had been shown previously to have a nutritional requirement for glycine. The enzyme was strongly inhibited by glycine and cysteine. The inhibition showed characteristics of allosteric inhibition and was pH-dependent. Inhibition by glycine, but not by cysteine, was highly specific. Analogues and derivatives of cysteine which contained a thiol group and a free amino group inhibited the activity of threonine aldolase. The presence of a carboxyl group was not necessary for inhibition. The cleavage of threonine by whole-cell suspensions was stimulated by either an energy source to aid transport or by rendering the cells permeable to substrate with oleate. Threonine did not appear to be degraded by enzymes other than threonine aldolase, as threonine dehydratase activity was low and NAD- and NADP-dependent threonine dehydrogenases were absent.     

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.     

On the Continuity of High Efficiency Modes in Linear RF Power Amplifiers

A novel formulation for the voltage waveforms in high efficiency linear power amplifiers is described. This formulation demonstrates that a constant optimum efficiency and output power can be obtained over a continuum of solutions by utilizing appropriate harmonic reactive impedance terminations. A specific example is confirmed experimentally. This new formulation has some important implications for the possibility of realizing broadband >10% high efficiency linear RF power amplifiers.