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Berlin Brandenburg

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  • Biochemistry  (50)
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  • 1
    Language: English
    In: Biochemistry, 14 January 2014, Vol.53(1), pp.20-9
    Description: Phytochromes constitute a class of photoreceptors that can be photoconverted between two stable states. The tetrapyrrole chromophore absorbs in the red spectral region and displays fluorescence maxima above 700 nm, albeit with low quantum yields. Because this wavelength region is particularly advantageous for fluorescence-based deep tissue imaging, there is a strong interest to engineer phytochrome variants with increased fluorescence yields. Such targeted design efforts would substantially benefit from a deeper understanding of those structural parameters that control the photophysical properties of the protein-bound chromophore. Here we have employed resonance Raman (RR) spectroscopy and molecular dynamics simulations for elucidating the chromophore structural changes in a fluorescence-optimized mutant (iRFP) derived from the PAS-GAF domain of the bacteriophytochrome RpBphP2 from Rhodopseudomas palustris . Both methods consistently reveal the structural consequences of the amino acid substitutions in the vicinity of the biliverdin chromophore that may account for lowering the propability of nonradiative excited state decays. First, compared to the wild-type protein, the tilt angle of the terminal ring D with respect to ring C is increased in iRFP, accompanied by the loss of hydrogen bond interactions of the ring D carbonyl function and the reduction of the number of water molecules in that part of the chromophore pocket. Second, the overall flexibility of the chromophore is significantly reduced, particularly in the region of rings D and A, thereby reducing the conformational heterogeneity of the methine bridge between rings A and B and the ring A carbonyl group, as concluded from the RR spectra of the wild-type proteins.
    Keywords: Phytochrome -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 2
    Language: English
    In: Biochemistry, 11 September 2012, Vol.51(36), pp.7040-2
    Description: Activation of the corrinoid [Fe-S] protein (CoFeSP), involved in reductive CO(2) conversion, requires the reduction of the Co(II) center by the [Fe-S] protein RACo, which according to the reduction potentials of the two proteins would correspond to an uphill electron transfer. In our resonance Raman spectroscopic work, we demonstrate that, as a conformational gate for the corrinoid reduction, complex formation of Co(II)FeSP and RACo specifically alters the structure of the corrinoid cofactor by modifying the interactions of the Co(II) center with the axial ligand. On the basis of various deletion mutants, the potential interaction domains on the partner proteins can be predicted.
    Keywords: Corrinoids -- Chemistry ; Iron-Sulfur Proteins -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 3
    Language: English
    In: Biochemistry, 23 July 2013, Vol.52(29), pp.4871-80
    Description: Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C-D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.
    Keywords: Anabaena -- Chemistry ; Phytochrome -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 4
    Language: English
    In: Biochemistry, 24 September 2013, Vol.52(38), pp.6601-14
    Description: The Ras converting enzyme (Rce1p) is an endoprotease that is involved in the post-translational processing of the Ras GTPases and other isoprenylated proteins. Its role in Ras biosynthesis marks Rce1p as an anticancer target. By assessing the chemical accessibility of cysteine residues substituted throughout the Saccharomyces cerevisiae Rce1p sequence, we have determined that yeast Rce1p has eight segments that are protected from chemical modification. Notably, the three residues that are essential for yeast Rce1p function (E156, H194, and H248) are all chemically inaccessible and associated with separate protected segments. By specifically assessing the chemical reactivity and glycosylation potential of the NH2 and COOH termini of Rce1p, we further demonstrate that Rce1p has an odd number of transmembrane spans. Substantial evidence that the most NH2-terminal segment functions as a transmembrane segment with the extreme NH2 terminus projecting into the endoplasmic reticulum (ER) lumen is presented. Because each of the remaining seven segments is too short to contain two spans and is flanked by chemically reactive positions, we infer that these segments are not transmembrane segments but rather represent compact structural features and/or hydrophobic loops that penetrate but do not fully span the bilayer (i.e., re-entrant helices). We thus propose a topological model in which yeast Rce1p contains a single transmembrane helix localized at its extreme NH2 terminus and one or more re-entrant helices and/or compact structural domains that populate the cytosolic face of the ER membrane. Lastly, we demonstrate that the natural cysteine residues of Rce1p are chemically inaccessible and fully dispensable for in vivo enzyme activity, formally eliminating the possibility of a cysteine-based enzymatic mechanism for this protease.
    Keywords: Cysteine -- Chemistry ; Metalloendopeptidases -- Chemistry ; Proprotein Convertases -- Chemistry ; Saccharomyces Cerevisiae Proteins -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 5
    Language: English
    In: Biochemistry, 31 July 2012, Vol.51(30), pp.5967-78
    Description: The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.
    Keywords: Cytochromes C -- Physiology ; Methionine -- Chemistry ; Saccharomyces Cerevisiae -- Enzymology ; Saccharomyces Cerevisiae Proteins -- Chemistry ; Tyrosine -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 6
    Language: English
    In: Biochemistry, 15 May 2018, Vol.57(19), pp.2747-2755
    Description: Recombinant production of heme proteins in Escherichia coli is often limited by the availability of heme in the host. Therefore, several methods, including the reconstitution of heme proteins after production but prior to purification or the HPEX system, conferring the ability to take up external heme have been developed and used in the past. Here we describe the use of the apathogenic E. coli strain Nissle 1917 (EcN) as a suitable host for the recombinant production of heme proteins. EcN has an advantage over commonly used lab strains in that it is able to take up heme from the environment through the heme receptor ChuA. Expression of several heme proteins from different prokaryotic sources led to high yield and quantitative incorporation of the cofactor when heme was supplied in the growth medium. Comparative UV-vis and resonance Raman measurements revealed that the method employed has significant influence on heme coordination with the EcN system representing the most native situation. Therefore, the use of EcN as a host for recombinant heme protein production represents an inexpensive and straightforward method to facilitate further investigations of structure and function.
    Keywords: Bacterial Outer Membrane Proteins -- Chemistry ; Escherichia Coli Proteins -- Chemistry ; Heme -- Chemistry ; Hemeproteins -- Chemistry ; Receptors, Cell Surface -- Chemistry ; Recombinant Proteins -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 7
    Language: English
    In: Biochemistry, 17 May 2016, Vol.55(19), pp.2722-34
    Description: Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.
    Keywords: Benzoquinones -- Chemistry ; Escherichia Coli -- Enzymology ; Escherichia Coli Proteins -- Chemistry ; Nad -- Chemistry ; Nadh Dehydrogenase -- Chemistry
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 8
    Language: English
    In: Biochemistry, May 17, 2016, Vol.55(19), pp.2722-2734
    Description: The article explores the substrateuprotein interaction in NDH-2 from Escherichia coli (EcNDH-2). It is suggested that type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD+ balance in cells. The presence of two distinct substrate binding sites for NADH and the quinine was demonstrated with the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes and it helped to identify a bound semiprotonated quinol as a catalytic intermediate.
    Keywords: Nicotinamide Adenine Dinucleotide – Analysis ; Escherichia Coli – Research ; Proteins – Analysis
    ISSN: 0006-2960
    E-ISSN: 1943295X
    Source: Cengage Learning, Inc.
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  • 9
    Language: English
    In: Biochemistry, 08 September 2015, Vol.54(35), pp.5389-400
    Description: Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named "light adaptation". The main objective of this spectroscopic study was to elucidate the molecular processes associated with light-dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all-trans configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all-trans,15-anti and 13-cis,15-syn configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)═C(14) and C(15)═N bonds. Both isomers of this DAapp state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)═C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DAapp) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-trans isomer than for the 13-cis isomer, possibly because of different chromophore-water interactions. Our data demonstrating two complementary photocycles of the DAapp isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light-dark adaptation, as suggested previously by electrical measurements.
    Keywords: Dark Adaptation -- Physiology ; Retinaldehyde -- Analogs & Derivatives
    ISSN: 00062960
    E-ISSN: 1520-4995
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  • 10
    Language: English
    In: Biochemistry, 29 September 2015, Vol.54(38), pp.5839-48
    Description: The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a green-absorbing state, Pg. Combining ultraviolet-vis (UV-vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.
    Keywords: Anabaena -- Metabolism ; Bacterial Proteins -- Metabolism ; Bile Pigments -- Metabolism ; Phycobilins -- Metabolism ; Phycocyanin -- Metabolism
    ISSN: 00062960
    E-ISSN: 1520-4995
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