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Article ; Online: Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins.

Leeder, Alexander J / Brown, Lynda J / Becker-Baldus, Johanna / Mehler, Michaela / Glaubitz, Clemens / Brown, Richard C D

Journal of labelled compounds & radiopharmaceuticals

2018 Volume 61, Issue 13, Page(s) 922–933

Abstract: Three all-trans retinals containing ... ...

Abstract	Three all-trans retinals containing multiple
MeSH term(s)	Chemistry Techniques, Synthetic ; Isotope Labeling ; Magnetic Resonance Spectroscopy/methods ; Membrane Proteins/chemistry ; Retinaldehyde/chemical synthesis ; Retinaldehyde/chemistry ; Stereoisomerism
Chemical Substances	Membrane Proteins ; Retinaldehyde (RR725D715M)
Language	English
Publishing date	2018-02-02
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	196095-7
ISSN	1099-1344 ; 0362-4803 ; 0022-2135
ISSN (online)	1099-1344
ISSN	0362-4803 ; 0022-2135
DOI	10.1002/jlcr.3576
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Article ; Online: Therapeutic drug monitoring of children and adolescents treated with aripiprazole: observational results from routine patient care.

Egberts, Karin / Reuter-Dang, Su-Yin / Fekete, Stefanie / Kulpok, Christine / Mehler-Wex, Claudia / Wewetzer, Christoph / Karwautz, Andreas / Mitterer, Michaela / Holtkamp, Kristian / Boege, Isabel / Burger, Rainer / Romanos, Marcel / Gerlach, Manfred / Taurines, Regina

Journal of neural transmission (Vienna, Austria : 1996)

2020 Volume 127, Issue 12, Page(s) 1663–1674

Abstract: Although aripiprazole is one of the most used antipsychotics, knowledge about serum concentrations in children and adolescents is scarce and age-specific therapeutic ranges have not been established yet. Data of a routine therapeutic drug monitoring ... ...

Abstract	Although aripiprazole is one of the most used antipsychotics, knowledge about serum concentrations in children and adolescents is scarce and age-specific therapeutic ranges have not been established yet. Data of a routine therapeutic drug monitoring service were analyzed in order to evaluate the relationship between dose and serum concentration of aripiprazole in children and adolescents. The study also aimed to evaluate whether the therapeutic reference range defined for adults with schizophrenia (100-350 ng/ml) is applicable for minors. Data from 130 patients (aged 7-19 years) treated with aripiprazole for different indications in doses of 2-30 mg/day were evaluated. Patient characteristics, doses, serum concentrations and therapeutic outcome were assessed by standardized measures. A positive mean correlation between body weight-corrected daily dose and aripiprazole concentration was found (r
MeSH term(s)	Adolescent ; Adult ; Antipsychotic Agents/therapeutic use ; Aripiprazole ; Child ; Drug Monitoring ; Female ; Humans ; Male ; Patient Care ; Schizophrenia/drug therapy
Chemical Substances	Antipsychotic Agents ; Aripiprazole (82VFR53I78)
Language	English
Publishing date	2020-09-30
Publishing country	Austria
Document type	Journal Article ; Observational Study ; Research Support, Non-U.S. Gov't
ZDB-ID	184163-4
ISSN	1435-1463 ; 0300-9564
ISSN (online)	1435-1463
ISSN	0300-9564
DOI	10.1007/s00702-020-02253-4
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Article ; Online: Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR.

Maciejko, Jakob / Mehler, Michaela / Kaur, Jagdeep / Lieblein, Tobias / Morgner, Nina / Ouari, Olivier / Tordo, Paul / Becker-Baldus, Johanna / Glaubitz, Clemens

Journal of the American Chemical Society

2015 Volume 137, Issue 28, Page(s) 9032–9043

Abstract: Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is ...

Abstract	Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.
MeSH term(s)	Amino Acid Sequence ; Bacterial Proteins/chemistry ; Lipid Bilayers/chemistry ; Models, Molecular ; Molecular Sequence Data ; Nuclear Magnetic Resonance, Biomolecular ; Protein Conformation ; Protein Multimerization ; Proteobacteria/chemistry ; Rhodopsins, Microbial/chemistry ; Salts/chemistry
Chemical Substances	Bacterial Proteins ; Lipid Bilayers ; Rhodopsins, Microbial ; Salts ; proteorhodopsin
Language	English
Publishing date	2015-07-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.5b03606
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Article ; Online: Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing.

Mehler, Michaela / Eckert, Carl Elias / Busche, Alena / Kulhei, Jennifer / Michaelis, Jonas / Becker-Baldus, Johanna / Wachtveitl, Josef / Dötsch, Volker / Glaubitz, Clemens

The Journal of biological chemistry

2015 Volume 290, Issue 46, Page(s) 27712–27722

Abstract: Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that ... ...

Abstract	Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.
MeSH term(s)	Inteins ; Kinetics ; Lipid Bilayers/chemistry ; Membrane Proteins/chemistry ; Nuclear Magnetic Resonance, Biomolecular ; Protein Engineering ; Protein Folding ; Protein Splicing ; Protein Structure, Secondary ; Rhodopsins, Microbial/chemistry
Chemical Substances	Lipid Bilayers ; Membrane Proteins ; Rhodopsins, Microbial ; proteorhodopsin
Language	English
Publishing date	2015-09-24
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M115.681205
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Article ; Online: Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR.

Mehler, Michaela / Eckert, Carl Elias / Leeder, Alexander J / Kaur, Jagdeep / Fischer, Tobias / Kubatova, Nina / Brown, Lynda J / Brown, Richard C D / Becker-Baldus, Johanna / Wachtveitl, Josef / Glaubitz, Clemens

Journal of the American Chemical Society

2017 Volume 139, Issue 45, Page(s) 16143–16153

Abstract: Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear ... ...

Abstract	Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating
MeSH term(s)	Nuclear Magnetic Resonance, Biomolecular/methods ; Proton Pumps/chemistry ; Proton Pumps/metabolism ; Proton Pumps/radiation effects ; Rhodopsins, Microbial/chemistry ; Rhodopsins, Microbial/metabolism ; Rhodopsins, Microbial/radiation effects ; Schiff Bases/chemistry
Chemical Substances	Proton Pumps ; Rhodopsins, Microbial ; Schiff Bases ; proteorhodopsin
Language	English
Publishing date	2017--15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/jacs.7b05061
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Article: Chromophore Distortions in Photointermediates of Proteorhodopsin Visualized by Dynamic Nuclear Polarization-Enhanced Solid-State NMR

Mehler, Michaela / Becker-Baldus Johanna / Brown Lynda J / Brown Richard C. D / Eckert Carl Elias / Fischer Tobias / Glaubitz Clemens / Kaur Jagdeep / Kubatova Nina / Leeder Alexander J / Wachtveitl Josef

Journal of the American Chemical Society. 2017 Nov. 15, v. 139, no. 45

2017

Abstract	Proteorhodopsin (PR) is the most abundant retinal protein on earth and functions as a light-driven proton pump. Despite extensive efforts, structural data for PR photointermediate states have not been obtained. On the basis of dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyze the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the ground state in comparison to light-induced, cryotrapped K- and M-states. A high M-state population could be achieved by preventing reprotonation of the Schiff base through a mutation of the primary proton donor (E108Q). Our data reveal unexpected large and alternating 13C chemical shift changes in the K-state propagating away from the Schiff base along the polyene chain. Furthermore, two different M-states have been observed reflecting the Schiff base reorientation after the deprotonation step. Our study provides novel insight into the photocycle of PR and also demonstrates the power of DNP-enhanced solid-state NMR to bridge the gap between functional and structural data and models.
Keywords	carbon ; models ; mutation ; nitrogen ; nuclear magnetic resonance spectroscopy ; proton pump ; rhodopsin ; schiff bases ; stable isotopes
Language	English
Dates of publication	2017-1115
Size	p. 16143-16153.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021%2Fjacs.7b05061
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Article: Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR

Maciejko, Jakob / Becker-Baldus Johanna / Glaubitz Clemens / Kaur Jagdeep / Lieblein Tobias / Mehler Michaela / Morgner Nina / Ouari Olivier / Tordo Paul

Journal of the American Chemical Society. 2015 July 22, v. 137, no. 28

2015

Abstract: Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of proteinâprotein interactions within the lipid bilayer ... ...

Abstract	Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of proteinâprotein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.
Keywords	lipid bilayers ; mass spectrometry ; membrane proteins ; nuclear magnetic resonance spectroscopy ; oligomerization ; polyacrylamide gel electrophoresis ; protein subunits ; protein-protein interactions ; rhodopsin
Language	English
Dates of publication	2015-0722
Size	p. 9032-9043.
Publishing place	American Chemical Society
Document type	Article
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021%2Fjacs.5b03606
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Article ; Online: Structural basis of the green-blue color switching in proteorhodopsin as determined by NMR spectroscopy.

Mao, Jiafei / Do, Nhu-Nguyen / Scholz, Frank / Reggie, Lenica / Mehler, Michaela / Lakatos, Andrea / Ong, Yean-Sin / Ullrich, Sandra J / Brown, Lynda J / Brown, Richard C D / Becker-Baldus, Johanna / Wachtveitl, Josef / Glaubitz, Clemens

Journal of the American Chemical Society

2014 Volume 136, Issue 50, Page(s) 17578–17590

Abstract: Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two ... ...

Abstract	Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green-blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14-C15. Upon mutation, a significantly stretched C14-C15 bond, deshielding of C15, and a slight alteration of the retinal chain's out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S0-S1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump-probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.
MeSH term(s)	Amino Acid Sequence ; Magnetic Resonance Spectroscopy ; Models, Molecular ; Molecular Sequence Data ; Mutation ; Rhodopsins, Microbial/chemistry ; Rhodopsins, Microbial/genetics ; Sequence Alignment
Chemical Substances	Rhodopsins, Microbial ; proteorhodopsin
Language	English
Publishing date	2014-12-17
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	3155-0
ISSN	1520-5126 ; 0002-7863
ISSN (online)	1520-5126
ISSN	0002-7863
DOI	10.1021/ja5097946
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Article ; Online: The EF loop in green proteorhodopsin affects conformation and photocycle dynamics.

Mehler, Michaela / Scholz, Frank / Ullrich, Sandra J / Mao, Jiafei / Braun, Markus / Brown, Lynda J / Brown, Richard C D / Fiedler, Sarah A / Becker-Baldus, Johanna / Wachtveitl, Josef / Glaubitz, Clemens

Biophysical journal

2013 Volume 105, Issue 2, Page(s) 385–397

Abstract: The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a ... ...

Abstract	The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced (13)C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.
MeSH term(s)	Amino Acid Sequence ; Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; Binding Sites ; Hydrogen-Ion Concentration ; Kinetics ; Light Signal Transduction ; Molecular Sequence Data ; Mutation ; Protein Structure, Tertiary ; Retinoids/chemistry ; Rhodopsin/chemistry ; Rhodopsin/genetics ; Rhodopsin/metabolism ; Rhodopsins, Microbial
Chemical Substances	Bacterial Proteins ; Retinoids ; Rhodopsins, Microbial ; proteorhodopsin ; retinylidene chromophore ; Rhodopsin (9009-81-8)
Language	English
Publishing date	2013-07-20
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2013.06.014
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Article: The EF Loop in Green Proteorhodopsin Affects Conformation and Photocycle dynamics

Mehler, Michaela / Scholz, Frank / Ullrich, Sandra J / Mao, Jiafei / Braun, Markus / Brown, Lynda J / Brown, Richard C.D / Fiedler, Sarah A / Becker-Baldus, Johanna / Wachtveitl, Josef / Glaubitz, Clemens

Biophysical journal. 2013 July 16, v. 105, no. 2

2013

Abstract	The proteorhodopsin family consists of retinal proteins of marine bacterial origin with optical properties adjusted to their local environments. For green proteorhodopsin, a highly specific mutation in the EF loop, A178R, has been found to cause a surprisingly large redshift of 20 nm despite its distance from the chromophore. Here, we analyze structural and functional consequences of this EF loop mutation by time-resolved optical spectroscopy and solid-state NMR. We found that the primary photoreaction and the formation of the K-like photo intermediate is almost pH-independent and slower compared to the wild-type, whereas the decay of the K-intermediate is accelerated, suggesting structural changes within the counterion complex upon mutation. The photocycle is significantly elongated mainly due to an enlarged lifetime of late photo intermediates. Multidimensional MAS-NMR reveals mutation-induced chemical shift changes propagating from the EF loop to the chromophore binding pocket, whereas dynamic nuclear polarization-enhanced 13C-double quantum MAS-NMR has been used to probe directly the retinylidene conformation. Our data show a modified interaction network between chromophore, Schiff base, and counterion complex explaining the altered optical and kinetic properties. In particular, the mutation-induced distorted structure in the EF loop weakens interactions, which help reorienting helix F during the reprotonation step explaining the slower photocycle. These data lead to the conclusion that the EF loop plays an important role in proton uptake from the cytoplasm but our data also reveal a clear interaction pathway between the EF loop and retinal binding pocket, which might be an evolutionary conserved communication pathway in retinal proteins.
Keywords	cytoplasm ; mutation ; nuclear magnetic resonance spectroscopy ; optical properties ; rhodopsin ; schiff bases
Language	English
Dates of publication	2013-0716
Size	p. 385-397.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2013.06.014
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