Untersuchungen zur Struktur, Funktion und Dynamik von Photosystem 2 aus dem thermophilen Cyanobakterium Thermosynechococcus elongatus\textit {Thermosynechococcus elongatus}

Ausgehend von einer neuen His-Tag PS2-Variante konnten durch den Einsatz sehr leistungsfähiger Chromatographietechniken verschiedene PS2-Subkomplexe in großen Mengen isoliert und in Kombination mit unterschiedlichen massenspektrometrischen Verfahren analysiert werden. Es konnte gezeigt werden, dass ein potenzielles PS2-Assemblierungsintermediat die bisher nicht näher charakterisierte Psb27 Untereinheit enthielt, bei welcher erstmals eine kovalente Lipidmodifikation nachgewiesen werden konnte. Ausgehend von Psb27 als Modellprotein konnten über einen bioinformatischen Ansatz weitere potenzielle Lipoproteine aus dem Bereich der Photosynthese in Cyanobakterien identifiziert werden. Darüber hinaus wurden mit der isolierten PsbO-Untereinheit ESR- und NMR-spektroskopische Untersuchungen durchgeführt, die neue Einblicke in die strukturelle Dynamik des Proteins ermöglichten. Zusätzlich konnten Informationen zur Interaktion der PsbO-Untereinheit mit potentiellen Liganden erhalten werden

Critical assessment of protein cross-linking and molecular docking

Photosystem II (PSII) is a large membrane-protein complex composed of about 20 subunits and various cofactors, which mediates the light-driven oxidation of water and reduction of plastoquinone, and is part of the photosynthetic electron transfer chain that is localized in the thylakoid membrane of cyanobacteria, algae, and plants. The stepwise assembly of PSII is guided and facilitated by numerous auxiliary proteins that play specific roles in this spatiotemporal process. Psb27, a small protein localized in the thylakoid lumen, appears to associate with an intermediate PSII complex that is involved in assembly of the $Mn_{4}CaO_{5}$ cluster. Its precise binding position on the PSII intermediate remains elusive, as previous approaches to the localization of Psb27 on PSII have yielded contradictory results. This was our motivation for a critical assessment of previously used methods and the development of an improved analysis pipeline. The combination of chemical cross-linking and mass spectrometry (CX-MS) with isotope-coded cross-linkers was refined and validated with reference to the PSII crystal structure. Psb27 was localized on the PSII surface adjacent to the large lumenal domain of CP43 on the basis of a cross-link connecting Psb27-K91 to CP43-K381. Additional contacts associating Psb27 with CP47 and the C-termini of D1 and D2 were detected by surface plasmon resonance (SPR) spectroscopy. This information was used to model the binding of Psb27 to the PSII surface in a region that is occupied by PsbV in the mature complex

The chloroplast SRP systems of Chaetosphaeridium globosum\textit {Chaetosphaeridium globosum} and Physcomitrella patens\textit {Physcomitrella patens} as intermediates in the evolution of SRP-dependent protein transport in higher plants

The bacterial signal recognition particle (SRP) mediates the cotranslational targeting of membrane proteins and is a high affinity complex consisting of a SRP54 protein subunit (Ffh) and an SRP RNA. The chloroplast SRP (cpSRP) pathway has adapted throughout evolution to enable the posttranslational targeting of the light harvesting chlorophyll a/b binding proteins (LHCPs) to the thylakoid membrane. In spermatophytes (seed plants), the cpSRP lacks the SRP RNA and is instead formed by a high affinity interaction of the conserved 54-kD subunit (cpSRP54) with the chloroplast-specific cpSRP43 protein. This heterodimeric cpSRP recognizes LHCP and delivers it to the thylakoid membrane. However, in contrast to spermatophytes, plastid SRP RNAs were identified within all streptophyte lineages and in all chlorophyte branches. Furthermore, it was shown that cpSRP43 does not interact with cpSRP54 in chlorophytes (e.g., $\textit {Chlamydomonas reinhardtii}$). In this study, we biochemically characterized the cpSRP system of the charophyte $\textit {Chaetosphaeridium globosum}$ and the bryophyte $\textit {Physcomitrella patens}$. Interaction studies demonstrate low affinity binding of cpSRP54 to cpSRP43 ($K_{d}$ ~10 $\mu$M) in $\textit {Chaetosphaeridium globosum}$ and $textit {Physcomitrella patens}$ as well as relatively low affinity binding of cpSRP54 to cpSRP RNA ($K_{d}$ ~1 $\mu$M) in $\textit {Physcomitrella patens}$. CpSRP54/cpSRP43 complex formation in charophytes is supported by the finding that specific alterations in the second chromodomain of cpSRP43, that are conserved within charophytes and absent in land plants, do not interfere with cpSRP54 binding. Furthermore, our data show that the elongated apical loop structure of the $\textit {Physcomitrella patens}$ cpSRP RNA contributes to the low binding affinity between cpSRP54 and the cpSRP RNA

Localization of the CyanoP binding site on photosystem II by surface plasmon resonance spectroscopy

Photosystem II (PSII), a large multi subunit membrane protein complex localized in the thylakoid membrane of cyanobacteria and chloroplasts, is the only known enzyme that catalyzes the light-driven oxidation of water. In addition to the membrane intrinsic part of PSII, efficient oxygen evolution requires soluble protein subunits at its luminal interface. In contrast to the detailed crystal structure of the active cyanobacterial complex the characterization of intermediate PSII species related to its assembly and repair is hampered by their instability or low abundance. As most structural variations of the corresponding PSII species are based on a different set of protein factors bound to the luminal interface of the complex we developed a system for interaction analysis between PSII and its soluble interaction partners based on surface plasmon resonance (SPR) spectroscopy. The assay was validated by the correct localization of the extrinsic PSII proteins PsbO, PsbV, and PsbU on the luminal PSII surface and used to determine the unknown binding position of CyanoP, the cyanobacterial homolog of higher plant PsbP. The CyanoP binding site was clearly localized in the center of PSII at a position, which is occupied by the PsbO subunit in mature PSII complexes. Consistently, we demonstrate selective binding of CyanoP to an inactive PSII assembly intermediate that lacks the extrinsic subunits PsbO, PsbV, and PsbU. These findings suggest, that CyanoP functions in the dynamic lifecycle of PSII, possibly in the association of CP$_47$ and CP$_43$ or in photoactivation of the oxygen-evolving complex

Scalable Fabrication of Biophotoelectrodes by Means of Automated Airbrush Spray-Coating

The fabrication and electrochemical evaluation of transparent photoelectrodes consisting of Photosystem I (PSI) or Photosystem II (PSII) is described, which are embedded and electrically wired by a redox polymer. The fabrication process is performed by an automated airbrush-type spray coating system, which ensures controlled and scalable electrode preparation. As proof of concept, electrodes with a surface area of up to 25 cm2 were prepared. The macro-porous structure of the indium tin oxide electrodes allows a high loading of the photoactive protein complexes leading to enhanced photocurrents, which are essential for potentially technologically relevant solar-powered devices. In addition, we show that unpurified crude PSII extracts, which can be provided in comparatively high yields for electrode modification, are suitable for photoelectrode fabrication with comparable photocurrent densities

Enzymatic oxyfunctionalization driven by photosynthetic water-splitting in the Cyanobacterium Synechocystis\it Synechocystis sp. PCC 6803

Photosynthetic water-splitting is a powerful force to drive selective redox reactions. The need of highly expensive redox partners such as NADPH and their regeneration is one of the main bottlenecks for the application of biocatalysis at an industrial scale. Recently, the possibility of using the photosystem of cyanobacteria to supply high amounts of reduced nicotinamide to a recombinant enoate reductase opened a new strategy for overcoming this hurdle. This paper presents the expansion of the photosynthetic regeneration system to a Baeyer-Villiger monooxygenase. Despite the potential of this strategy, this work also presents some of the encountered challenges as well as possible solutions, which will require further investigation. The successful enzymatic oxygenation shows that cyanobacterial whole-cell biocatalysis is an applicable approach that allows fuelling selective oxyfunctionalisation reactions at the expense of light and water. Yet, several hurdles such as side-reactions and the cell-density limitation, probably due to self-shading of the cells, will have to be overcome on the way to synthetic applications

The 5 kDa protein NdhP is essential for stable NDH-1L assembly in Thermosynechococcus elongatus\textit {Thermosynechococcus elongatus}

The cyanobacterial NADPH:plastoquinone oxidoreductase complex (NDH-1), that is related to Complex I of eubacteria and mitochondria, plays a pivotal role in respiration as well as in cyclic electron transfer (CET) around PSI and is involved in a unique carbon concentration mechanism (CCM). Despite many achievements in the past, the complex protein composition and the specific function of many subunits of the different NDH-1 species remain elusive. We have recently discovered in a NDH-1 preparation from $\textit {Thermosynechococcus elongatus}$ two novel single transmembrane peptides (NdhP, NdhQ) with molecular weights below 5 kDa. Here we show that NdhP is a unique component of the ~450 kDa NDH-1L complex, that is involved in respiration and CET at high $CO_{2}$ concentration, and not detectable in the NDH-1MS and NDH-1MS' complexes that play a role in carbon concentration. C-terminal fusion of NdhP with his-tagged superfolder GFP and the subsequent analysis of the purified complex by electron microscopy and single particle averaging revealed its localization in the NDH-1L specific distal unit of the NDH-1 complex, that is formed by the subunits NdhD1 and NdhF1. Moreover, NdhP is essential for NDH-1L formation, as this type of NDH-1 was not detectable in a $\Delta$$\it ndhP$::Km mutant

Closing the gap for electronic short-circuiting

Well-defined assemblies of photosynthetic protein complexes are required for an optimal performance of semi-artificial energy conversion devices, capable of providing unidirectional electron flow when light-harvesting proteins are interfaced with electrode surfaces. We present mixed photosystem I (PSI) monolayers constituted of native cyanobacterial PSI trimers in combination with isolated PSI monomers from the same organism. The resulting compact arrangement ensures a high density of photoactive protein complexes per unit area, providing the basis to effectively minimize short-circuiting processes that typically limit the performance of PSI-based bioelectrodes. The PSI film is further interfaced with redox polymers for optimal electron transfer, enabling highly efficient light-induced photocurrent generation. Coupling of the photocathode with a [NiFeSe]-hydrogenase confirms the possibility to realize light-induced H$_2$ evolution

Infantile restrictive cardiomyopathy

$\it TNNI3$ encoding cTnI, the inhibitory subunit of the troponin complex, is the main target for mutations leading to restrictive cardiomyopathy (RCM). Here we investigate two cTnI-R170G/W amino acid replacements, identified in infantile RCM patients, which are located in the regulatory C-terminus of cTnI. The C-terminus is thought to modulate the function of the inhibitory region of cTnI. Both cTnI-R170G/W strongly enhanced the $Ca^{2+}$-sensitivity of skinned fibres, as is typical for RCM-mutations. Both mutants strongly enhanced the affinity of troponin (cTn) to tropomyosin compared to wildtype cTn, whereas binding to actin was either strengthened (R170G) or weakened (R170W). Furthermore, the stability of reconstituted thin filaments was reduced as revealed by electron microscopy. Filaments containing R170G/W appeared wavy and showed breaks. Decoration of filaments with myosin subfragment S1 was normal in the presence of R170W, but was irregular with R170G. Surprisingly, both mutants did not affect the $Ca^{2+}$-dependent activation of reconstituted cardiac thin filaments. In the presence of the N-terminal fragment of cardiac myosin binding protein C (cMyBPC-C0C2) cooperativity of thin filament activation was increased only when the filaments contained wildtype cTn. No effect was observed in the presence of cTn containing R170G/W. cMyBPC-C0C2 significantly reduced binding of wildtype troponin to actin/tropomyosin, but not of both mutant cTn. Moreover, we found a direct troponin/cMyBPC-C0C2 interaction using microscale thermophoresis and identified cTnI and cTnT, but not cTnC as binding partners for cMyBPC-C0C2. Only cTn containing cTnI-R170G showed a reduced affinity towards cMyBPC-C0C2. Our results suggest that the RCM cTnI variants R170G/W impair the communication between thin and thick filament proteins and destabilize thin filaments

De novo missense mutations in TNNC1\it TNNC1 and TNNI3\it TNNI3 causing severe infantile cardiomyopathy affect myofilament structure and function and are modulated by troponin targeting agents

Rare pediatric non-compaction and restrictive cardiomyopathy are usually associated with a rapid and severe disease progression. While the non-compaction phenotype is characterized by structural defects and is correlated with systolic dysfunction, the restrictive phenotype exhibits diastolic dysfunction. The molecular mechanisms are poorly understood. Target genes encode among others, the cardiac troponin subunits forming the main regulatory protein complex of the thin filament for muscle contraction. Here, we compare the molecular effects of two infantile de novo point mutations in $\it TNNC1$ ($\it {p.cTnC-G34S}$) and $\it TNNI3$ (($\it {p.cTnI-D127Y}$) leading to severe non-compaction and restrictive phenotypes, respectively. We used skinned cardiomyocytes, skinned fibers, and reconstituted thin filaments to measure the impact of the mutations on contractile function. We investigated the interaction of these troponin variants with actin and their inter-subunit inter-actions, as well as the structural integrity of reconstituted thin filaments. Both mutations exhibited similar functional and structural impairments, though the patients developed different phenotypes. Furthermore, the protein quality control system was affected, as shown for TnC-G34S using patient’s myocardial tissue samples. The two troponin targeting agents levosimendan and green tea extract (-)-epigallocatechin-3-gallate (EGCg) stabilized the structural integrity of reconstituted thin filaments and ameliorated contractile function in vitro in some, but not all, aspects to a similar degree for both mutations