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Berlin Brandenburg

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  • 1
    Language: English
    In: Journal of the American Chemical Society, 21 March 2018, Vol.140(11), pp.4085-4091
    Description: Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the M intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. N(SB)-H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and C-C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, N-C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.
    Keywords: Light ; Nuclear Magnetic Resonance, Biomolecular ; Bacteriorhodopsins -- Chemistry ; Ion Pumps -- Chemistry
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 2
    Language: English
    In: Biophysical Journal, 22 September 2010, Vol.99(6), pp.1932-1939
    Description: Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil- - - - -coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
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  • 3
    Language: English
    In: Origins of Life and Evolution of Biospheres, 2011, Vol.41(1), pp.17-22
    Description: Reactions of short sugars under mild, plausibly prebiotic conditions yield organic microspherules that may have played a role in prebiotic chemistry as primitive reaction vessels. It has been widely thought that nitrogen chemistry, in particular Amadori rearrangement, is central to this process, Here we show that microspherules form in the absence of any nitrogen compounds if the pH is sufficiently low. In particular, while the microspherule formation induced by ammonium acetate (pH 7) is not reproduced by ammonium chloride (pH 5), it is reproduced by oxalic acid and by hydrochloric acid (pH 1). The formation of microspherules in the presence of oxalic acid is similar to that in the presence of ammonium acetate: aqueous reactions of D-erythrose, D-ribose, 2-deoxy-D-ribose and D-fructose in the presence of oxalic acid produce microspherules ranging in size from approximately 1–5 μm after eight weeks incubation at 65°C, while the aldohexoses D-glucose, D-galactose and D-mannose do not. This pattern correlates with the occurrence of furanose forms in these sugars.
    Keywords: Prebiotic chemistry ; Sugar browning ; Sugar polymerization ; Amadori ; Maillard
    ISSN: 0169-6149
    E-ISSN: 1573-0875
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  • 4
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 09 June 2009, Vol.106(23), pp.9244-9
    Description: Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90. Multidimensional spectroscopy of U-(13)C,(15)N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.
    Keywords: Bacteriorhodopsins -- Chemistry ; Nuclear Magnetic Resonance, Biomolecular -- Methods
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 5
    Language: English
    In: Biochemistry, 14 May 2002, Vol.41(19), pp.6026-31
    Description: Light-driven proton transport in bacteriorhodopsin (BR) is initiated by photoisomerization of the retinylidene chromophore, which perturbs the hydrogen bonding network in the Schiff base region of the active site. This study aimed to identify the frequency and dipolar orientation of the N-D stretching vibrations of the Schiff base before and after photoisomerization, by means of low-temperature polarized FTIR spectroscopy of [zeta-(15)N]lysine-labeled BR in D(2)O. (15)N-shifted modes were found at 2123 and 2173 cm(-1) for BR, and at 2468 and 2495 cm(-1) for the K intermediate. The corresponding N-H stretches are at approximately 2800 cm(-1) for BR and 3350-3310 cm(-1) for the K intermediate. The shift to a 350 cm(-1) higher frequency upon photoisomerization is consistent with loss of the hydrogen bond of the Schiff base. The N-D stretch frequencies of the Schiff base in BR and the K intermediate are close to the O-D stretch frequencies of strongly hydrogen bonded water and Thr89, respectively. The angles of the dipole moments of the N-D stretches to the membrane normal were determined to be 60-65 degrees for BR and approximately 90 degrees for the K intermediate. In the case of BR, the stretch orientation is expected to deviate from the N-D bond orientation due to vibrational mixing in the hydrogen bonding network. In contrast, the data for the K intermediate suggest that the N-D group is not hydrogen bonded and orients along the membrane.
    Keywords: Bacteriorhodopsins -- Chemistry
    ISSN: 0006-2960
    E-ISSN: 15204995
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  • 6
    Language: English
    In: Journal of the American Chemical Society, Dec 26, 2001, Vol.123(51), p.12929-12930
    Description: Application of frequency-selective REDOR is demonstrated to distance measurements between Schiff base and aspartic acids in the active sites of uniformly (super 13)C, (super15)N-labeled bacteriorhodopsin. The measured distances are in agreement with the diffraction structures of light-adapted bR and NMR methods.
    Keywords: Carbon Compounds -- Structure ; Carbon Compounds -- Research ; Nuclear Magnetic Resonance -- Research ; Bacteriorhodopsin -- Research
    ISSN: 0002-7863
    E-ISSN: 15205126
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  • 7
    Language: English
    In: Journal of Biomolecular NMR, 2013, Vol.57(2), pp.129-139
    Description: The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1- 13 C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2- 13 C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.
    Keywords: Isotope substitution ; Sparse labeling ; MAS NMR structure determination ; Dipolar truncation ; Isotope labeled peptone ; C-acetate
    ISSN: 0925-2738
    E-ISSN: 1573-5001
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  • 8
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 13 February 2001, Vol.98(4), pp.1571-1576
    Description: Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O-H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3- 18 O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O-D group of Thr-89 in D 2O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O-H (O-D) group of Thr-89 in M is indicated by a shift in the C=O stretching vibration of the former because of 18 O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.
    Keywords: Physical sciences -- Chemistry -- Chemical bonding ; Physical sciences -- Physics -- Matter ; Physical sciences -- Chemistry -- Chemical elements ; Physical sciences -- Chemistry -- Chemical compounds ; Physical sciences -- Physics -- Microphysics ; Applied sciences -- Laboratory techniques -- Spectroscopy ; Physical sciences -- Physics -- Microphysics ; Physical sciences -- Physics -- Microphysics ; Physical sciences -- Physics -- Mechanics ; Physical sciences -- Chemistry -- Chemical reactions
    ISSN: 00278424
    E-ISSN: 10916490
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  • 9
    Language: English
    In: Journal of Molecular Microbiology and Biotechnology, August 2013, Vol.23(4-5), pp.281-289
    Description: The buoyancy organelles of aquatic microorganisms have to meet stringent specifications: allowing gases to equilibrate freely across the proteinaceous shell, preventing the condensation of water vapor inside the hollow cavity and resisting collapse under hydrostatic pressures that vary with column depth. These properties are provided by the 7- to 8-kDa gas vesicle protein A (GvpA), repeats of which form all but small, specialized portions of the shell. Magic angle spinning nuclear magnetic resonance is uniquely capable of providing high-resolution information on the fold and assembly of GvpA. Here we compare results for the gas vesicles of the haloarchaea Halobacterium salinarum with those obtained previously for the cyanobacterium Anabaena flos-aquae. The data suggest that the two organisms follow similar strategies for avoiding water condensation. On the other hand, in its relatively shallow habitat, H. salinarum is able to avoid collapse with a less costly GvpA fold than is adopted by A. flos-aquae.
    Keywords: Protein Membrane-Enclosed Organelles ; Protein Structure ; Chemical Shift Assignment ; Cyanobacteria ; Anabaena ; Haloarchaea ; Halobacterium ; Gas Vesicle Protein A ; Amyloid ; Supramolecular Assembly ; Biology
    ISBN: 9783318024548
    ISBN: 3318024546
    ISSN: 1464-1801
    E-ISSN: 1660-2412
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  • 10
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 22 January 2008, Vol.105(3), pp.883-8
    Description: By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The (15)N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.
    Keywords: Bacteriorhodopsins -- Chemistry
    ISSN: 00278424
    E-ISSN: 1091-6490
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