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Linking protistan community shifts along salinity gradients with cellular haloadaptation strategies
(2019)
Salinity is one of the most structuring environmental factors for microeukaryotic communities. Using eDNA barcoding, I detected significant shifts in microeukaryotic community compositions occurring at distinct salinities between brackish and marine conditions in the Baltic Sea. I, furthermore, conducted a metadata analysis including my and other marine and hypersaline community sequence data to confirm the existence of salinity-related transition boundaries and significant changes in alpha diversity patterns along a brackish to hypersaline gradient. One hypothesis for the formation of salinity-dependent transition boundaries between brackish to hypersaline conditions is the use of different cellular haloadaptation strategies. To test this hypothesis, I conducted metatranscriptome analyses of microeukaryotic communities along a pronounced salinity gradient (40 – 380 ‰). Clustering of functional transcripts revealed differences in metabolic properties and metabolic capacities between microeukaryotic communities at specific salinities, corresponding to the transition boundaries already observed in the taxonomic eDNA barcoding approach. In specific, microeukaryotic communities thriving at mid-hypersaline conditions (≤ 150 ‰) seem to predominantly apply the ‘low-salt – organic-solutes-in’ strategy by accumulating compatible solutes to counteract osmotic stress. Indications were found for both the intracellular synthesis of compatible solutes as well as for cellular transport systems. In contrast, communities of extreme-hypersaline habitats (≥ 200 ‰) may preferentially use the ‘high-salt-in’ strategy, i. e. the intracellular accumulation of inorganic ions in high concentrations, which is implied by the increased expression of Mg2+, K+, Cl- transporters and channels.
In order to characterize the ‘low-salt – organic-solutes-in’ strategy applied by protists in more detail, I conducted a time-resolved transcriptome analysis of the heterotrophic ciliate Schmidingerothrix salinarum serving as model organism. S. salinarum was thus subjected to a salt-up shock to investigate the intracellular response to osmotic stress by shifts of gene expression. After increasing the external salinity, an increased expression of two-component signal transduction systems and MAPK cascades was observed. In an early reaction, the expression of transport mechanisms for K+, Cl- and Ca2+ increased, which may enhance the capacity of K+, Cl- and Ca2+ in the cytoplasm to compensate possibly harmful Na+ influx. Expression of enzymes for the synthesis of possible compatible solutes, starting with glycine betaine, followed by ectoine and later proline, could imply that the inorganic ions K+, Cl- and Ca2+ are gradually replaced by the synthesized compatible solutes. Additionally, expressed transporters for choline (precursor of glycine betaine) and proline could indicate an intracellular accumulation of compatible solutes to balance the external salinity. During this accumulation, the up-regulated ion export mechanisms may increase the capacity for Na+ expulsion from the cytoplasm and ion compartmentalization between cell organelles seem to happen.
The results of my PhD project revealed first evidence at molecular level for the salinity-dependent use of different haloadaptation strategies in microeukaryotes and significantly extend existing knowledge about haloadaptation processes in ciliates. The results provide ground for future research, such as (comparative) transcriptome analysis of ciliates thriving in extreme-hypersaline habitats or experiments like qRT-PCR to validate transcriptome results.
Function of two redox sensing kinases from the methanogenic archaeon Methanosarcina acetivorans
(2019)
MsmS is a heme-based redox sensor kinase in Methanosarcina acetivorans consisting of alternating PAS and GAF domains connected to a C-terminal kinase domain. In addition to MsmS, M. acetivorans possesses a second kinase, MA0863 with high sequence similarity. Interestingly, MA0863 possesses an amber codon in its second GAF domain, encoding for the amino acid pyrrolysine. Thus far, no function of this residue has been resolved. In order to examine the heme iron coordination in both proteins, an improved method for the production of heme proteins was established using the Escherichia coli strain Nissle 1917. This method enables the complete reconstitution of a recombinant hemoprotein during protein production, thereby resulting in a native heme coordination. Analysis of the full-length MsmS and MA0863 confirmed a covalently bound heme cofactor, which is connected to one conserved cysteine residue in each protein. In order to identify the coordinating amino acid residues of the heme iron, UV/vis spectra of different variants were measured. These studies revealed His702 in MsmS and the corresponding His666 in MA0863 as the proximal heme ligands. MsmS has previously been described as a heme-based redox sensor. In order to examine whether the same is true for MA0863, redox dependent kinase assays were performed. MA0863 indeed displays redox dependent autophosphorylation activity, which is independent of heme ligands and only observed under oxidizing conditions. Interestingly, autophosphorylation was shown to be independent of the heme cofactor but rather relies on thiol oxidation. Therefore, MA0863 was renamed in RdmS (redox dependent methyltransferase-associated sensor). In order to identify the phosphorylation site of RdmS, thin layer chromatography was performed identifying a tyrosine as the putative phosphorylation site. This observation is in agreement with the lack of a so-called H-box in typical histidine kinases. Due to their genomic localization, MsmS and RdmS were postulated to form two-component systems (TCS) with vicinal encoded regulator proteins MsrG and MsrF. Therefore, protein-protein interaction studies using the bacterial adenylate two hybrid system were performed suggesting an interaction of RdmS and MsmS with the three regulators MsrG/F/C. Due to these multiple interactions these signal transduction pathways should rather be considered multicomponent system instead of two component systems.
The transfer of substrates between to enzymes within a biosynthesis pathway is an effective way to synthesize the specific product and a good way to avoid metabolic interference. This process is called metabolic channeling and it describes the (in-)direct transfer of an intermediate molecule between the active sites of two enzymes. By forming multi-enzyme cascades the efficiency of product formation and the flux is elevated and intermediate products are transferred and converted in a correct manner by the enzymes.
During tetrapyrrole biosynthesis several substrate transfer events occur and are prerequisite for an optimal pigment synthesis. In this project the metabolic channeling process during the pink pigment phycoerythrobilin (PEB) was investigated. The responsible ferredoxin-dependent bilin reductases (FDBR) for PEB formation are PebA and PebB. During the pigment synthesis the intermediate molecule 15,16-dihydrobiliverdin (DHBV) is formed and transferred from PebA to PebB. While in earlier studies a metabolic channeling of DHBV was postulated, this work revealed new insights into the requirements of this protein-protein interaction. It became clear, that the most important requirement for the PebA/PebB interaction is based on the affinity to their substrate/product DHBV. The already high affinity of both enzymes to each other is enhanced in the presence of DHBV in the binding pocket of PebA which leads to a rapid transfer to the subsequent enzyme PebB. DHBV is a labile molecule and needs to be rapidly channeled in order to get correctly further reduced to PEB. Fluorescence titration experiments and transfer assays confirmed the enhancement effect of DHBV for its own transfer.
More insights became clear by creating an active fusion protein of PebA and PebB and comparing its reaction mechanism with standard FDBRs. This fusion protein was able to convert biliverdin IXα (BV IXα) to PEB similar to the PebS activity, which also can convert BV IXα via DHBV to PEB as a single enzyme. The product and intermediate of the reaction were identified via HPLC and UV-Vis spectroscopy.
The results of this work revealed that PebA and PebB interact via a proximity channeling process where the intermediate DHBV plays an important role for the interaction. It also highlights the importance of substrate channeling in the synthesis of PEB to optimize the flux of intermediates through this metabolic pathway.
The screening of metagenomic datasets led to the identification of new phage-derived members of the heme oxygenase and the ferredoxin-dependent bilin reductase enzyme families.
The novel bilin biosynthesis genes were shown to form mini-cassettes on metagenomic scaffolds and further form distinct clusters in phylogenetic analyses (Ledermann et al., 2016). In this project, it was demonstrated that the discovered sequences actually encode for active enzymes. The biochemical characterization of a member of the heme oxygenases (ΦHemO) revealed that it possesses a regiospecificity for the α-methine bridge in the cleavage of the heme macrocycle. The reaction product biliverdin IXα was shown to function as the substrate for the novel ferredoxin-dependent bilin reductases (PcyX reductases), which catalyze its reduction to PEB via the intermediate 15,16-DHBV. While it was demonstrated that ΦPcyX, a phage-derived member of the PcyX reductases, is an active enzyme, it also became clear that the rate of the reaction is highly dependent on the employed redox partner. It turned out that the ferredoxin from the cyanophage P-SSM2 is to date the most suitable redox partner for the reductases of the PcyX group. Furthermore, the solution of the ΦPcyX crystal structure revealed that it adopts an α/β/α-sandwich fold, typical for the FDBR-family. Activity assays and subsequent HPLC analyses with different variants of the ΦPcyX protein demonstrated that, despite their similarity, PcyX and PcyA reductases must act via different reaction mechanisms.
Another part of this project focused on the biochemical characterization of the FDBR KflaHY2 from the streptophyte alga Klebsormidium flaccidum. Experiments with recombinant KflaHY2 showed that it is an active FDBR which produces 3(Z)-PCB as the main reaction product, like it can be found in reductases of the PcyA group. Moreover, it was shown that under the employed assay conditions the reaction of BV to PCB proceeds in two different ways: Both 3(Z)-PΦB and 18¹,18²-DHBV occur as intermediates. Activity assays with the purified intermediates yielded PCB. Hence, both compounds are suitable substrates for KflaHY2.
The results of this work highlight the importance of the biochemical experiments, as catalytic activity cannot solely be predicted by sequence analysis.
Grape powdery mildew, Erysiphe necator, is one of the most significant plant pathogens, which affects grape growing regions world-wide. Because of its short generation time and the production of large amounts of conidia throughout the season, E. necator is classified as a moderate to high risk pathogen with respect to the development of fungicide resistance. The number of fungicidal mode of actions available to control powdery mildew is limited and for some of them resistances are already known. Aryl-phenyl-ketones (APKs), represented by metrafenone and pyriofenone, and succinate-dehydrogenase inhibitors (SDHIs), composed of numerous active ingredients, are two important fungicide classes used for the control of E. necator. Over the period 2014 to 2016, the emergence and development of metrafenone and SDHI resistant E. necator isolates in Europe was followed and evaluated. The distribution of resistant isolates was thereby strongly dependent on the European region. Whereas the north-western part is still predominantly sensitive, samples from east European countries showed higher resistance frequencies.
Classical sensitivity tests with obligate biotrophs can be challenging regarding sampling, transport and especially the maintenance of the living strains. Whenever possible, molecular genetic methods are preferred for a more efficient monitoring. Such methods require the knowledge of the resistance mechanisms. The exact molecular target and the resistance mechanism of metrafenone is still unknown. Whole genome sequencing of metrafenone sensitive and resistant wheat powdery mildew isolates, as well as adapted laboratory mutants of Aspergillus nidulans, where performed with the aim to identify proteins potentially linked to the mode of action or which contribute to metrafenone resistance. Based on comparative SNP analysis, four proteins potentially associated with metrafenone resistance were identified, but validation studies could not confirm their role in metrafenone resistance. In contrast to APKs, the mode of action of SDHIs is well understood. Sequencing of the sdh-genes of less sensitive E. necator isolates identified four different target-site mutations, the B-H242R, B-I244V, C-G169D and C-G169S, in sdhB and sdhC, respectively. Based on this information it was possible to develop molecular genetic monitoring methods for the mutations B-H242R and C-G169D. In 2016, the B-H242R was thereby identified as by far the most frequent mutation. Depending on the analysed SDH compound and the sdh-genotype, different sensitivities were observed and revealed a complex cross-resistance pattern.
Growth competition assays without selection pressure, with mixtures of sensitive and resistant E. necator isolates, were performed to determine potential fitness costs associated with fungicide resistance. With the experimental setups used, a clear fitness disadvantage associated with metrafenone resistance was not identified, although a strong variability of fitness was observed among the tested resistant E. necator isolates. For isolates with a reduced sensitivity towards SDHIs, associated fitness costs were dependent on the sdh-genotype analysed. Competition tests with the B-H242R genotypes gave evidence that there are no fitness costs associated with this mutation. In contrast, the C-G169D genotypes were less competitive, indicating a restricted fitness compared to the tested sensitive partners. Competition assays of field isolates, which exhibited several resistances towards different fungicide classes, indicated that there are no fitness costs associated with a multiple resistant phenotype in E. necator. Overall, these results clearly indicate the importance to analyse a representative number of isolates with sensitive and resistant phenotypes.
The cytosolic Fe65 adaptor protein family, consisting of Fe65, Fe65L1 and Fe65L2 is involved in many intracellular signaling pathways linking via its three interaction domains a continuously growing list of proteins by facilitating functional interactions. One of the most important binding partners of Fe65 family proteins is the amyloid precursor protein (APP), which plays an important role in Alzheimer Disease.
To gain deeper insights in the function of the ubiquitously expressed Fe65 and the brain enriched Fe65L1, the goal of my study was I) to analyze their putative synaptic function in vivo, II) to examine structural analysis focusing on a putative dimeric complex of Fe65, III) to consider the involvement of Fe65 in mediating LRP1 and APP intracellular trafficking in murine hippocampal neurons. By utilizing several behavioral analyses of Fe65 KO, Fe65L1 KO and Fe65/Fe65L1 DKO mice I could demonstrate that the Fe65 protein family is essential for learning and memory as well as grip strength and locomotor activity. Furthermore, immunohistological as well as protein biochemical analysis revealed that the Fe65 protein family is important for neuromuscular junction formation in the peripheral nervous system, which involves binding of APP and acting downstream of the APP signaling pathway. Via Co-immunoprecipitation analysis I could verify that Fe65 is capable to form dimers ex vivo, which exclusively occur in the cytosol and upon APP expression are shifted to membrane compartments forming trimeric complexes. The influence of the loss of Fe65 and/or Fe65L1 on APP and/or LRP1 transport characteristics in axons could not be verified, possibly conditioned by the compensatory effect of Fe65L2. However, I could demonstrate that LRP1 affects the APP transport independently of Fe65 by shifting APP into slower types of vesicles leading to changed processing and endocytosis of APP.
The outcome of my thesis advanced our understanding of the Fe65 protein family, especially its interplay with APP physiological function in synapse formation and synaptic plasticity.
The biodiversity of the cyanobacterial lichen flora of Vietnam is chronically understudied. Previous studies often neglected the lichens that inhabit lowlands especially outcrops and sand dunes that are common habitats in Vietnam.
A cyanolichen collection was gathered from lowlands of central and southern Vietnam to study their diversity and distribution. At the same time, cultured photobionts from those lichens were used for olyphasic taxonomic approach.
A total of 66 cyanolichens were recorded from lowland regions in central and southern of Vietnam, doubles the number of cyanolichens for Vietnam. 80% of them are new records for Vietnam in which a new species Pyrenopsis melanophthalma and two new unidentified lichinacean taxa were described.
A notably floristic segregation by habitats was indicated in the communities. Saxicolous Lichinales dominated in coastal outcrops that corresponded to 56% of lichen species richness. Lecanoralean cyanolichens and basidiolichens were found in the lowland forests. Precipitation correlated negatively to species richness in this study, indicating a competitive relationship.
Eleven cyanobacterial strains including 8 baeocyte-forming members of the genus Chroococcidiopsis and 3 heterocyte-forming species of the genera Nostoc and Scytonema were successfully isolated from lichens.
Phylogenetic and morphological analyses indicated that Chroococcidiopsis was the unique photobiont in Peltula. New mophological characters were found in two Chroococcidiopsis strains: (1) the purple content of cells in one photobiont strain that was isolated from a new lichinacean taxon, and (2) the pseudofilamentous feature by binary division from a strain that was isolated from Porocyphus dimorphus.
With respect to heterocyte-forming cyanobiont, Scytonema was confirmed as the photobiont in the ascolichen Heppia lutosa applying the polyphasic method. The genus Scytonema in the basidiolichens Cyphellostereum was morphologically examinated in lichen thalli. For the first time the intracellular haustorial system of basidiolichen genus Cyphellostereum was noted and investigated.
Phylogenetic analysis of photobiont strains Nostoc from Pannaria tavaresii and Parmeliella brisbanensis indicated that a high selectivity occurred in Parmeliella brisbanensis that were from different regions of the world, while low photobiont selectivity occurred among Pannaria tavaresii samples from different geographical regions.
The herewith presented dissertation is therefore an important contribution to the lichen flora of Vietnam and a significant improvement of the actual knowledge about cyanolichens in this country.
Human forest modification is among the largest global drivers of terrestrial degradation
of biodiversity, species interactions, and ecosystem functioning. One of the most
pertinent components, forest fragmentation, has a long history in ecological research
across the globe, particularly in lower latitudes. However, we still know little how
fragmentation shapes temperate ecosystems, irrespective of the ancient status quo of
European deforestation. Furthermore, its interaction with another pivotal component
of European forests, silvicultural management, are practically unexplored. Hence,
answering the question how anthropogenic modification of temperate forests affects
fundamental components of forest ecosystems is essential basic research that has
been neglected thus far. Most basal ecosystem elements are plants and their insect
herbivores, as they form the energetic basis of the tropic pyramid. Furthermore, their
respective biodiversity, functional traits, and the networks of interactions they
establish are key for a multitude of ecosystem functions, not least ecosystem stability.
Hence, the thesis at hand aimed to disentangle this complex system of
interdependencies of human impacts, biodiversity, species traits and inter-species
interactions.
The first step lay in understanding how woody plant assemblages are shaped by
human forest modification. For this purpose, field investigations in 57 plots in the
hyperfragmented cultural landscape of the Northern Palatinate highlands (SW
Germany) were conducted, censusing > 4,000 tree/shrub individuals from 34 species.
Use of novel, integrative indices for different types of land-use allowed an accurate
quantification of biotic responses. Intriguingly, woody tree/shrub communities reacted
strikingly positive to forest fragmentation, with increases in alpha and beta diversity,
as well as proliferation of heat/drought/light adapted pioneer species. Contrarily,
managed interior forests were homogenized/constrained in biodiversity, with
dominance of shade/cold adapted commercial tree species. Comparisons with recently
unmanaged stands (> 40 a) revealed first indications for nascent conversion to oldgrowth
conditions, with larger variability in light conditions and subsequent
community composition. Reactions to microclimatic conditions, the relationship
between associated species traits and the corresponding species pool, as well as
facilitative/constraining effects by foresters were discussed as underlying mechanisms.
Reactions of herbivore assemblages to forest fragmentation and the subsequent
changes in host plant communities were assessed by comprehensive sampling of >
1,000 live herbivores from 134 species in the forest understory. Diversity was –
similarly to plant communities - higher in fragmentation affected habitats, particularly
in edges of continuous control forests. Furthermore, average trophic specialization
showed an identical pattern. Mechanistically, benefits from microclimatic conditions,
host availability, as well as pronounced niche differentiation are deemed responsible.
While communities were heterogeneous, with no segregation across habitats, (smallforest fragments, edges, and interior of control forests), vegetation diversity, herbivore
diversity, as well as trophic specialization were identified to shape community
composition. This probably reflected a gradient from generalistic/species poor vs.
specialist/species rich herbivore assemblages.
Insect studies conducted in forest systems are doomed to incompleteness
without considering ‘the last biological frontier’, the tree canopies. To access their
biodiversity, relationship to edge effects, and their conservational value, the
arboricolous arthropod fauna of 24 beech (Fagus sylvatica) canopies was sampled via
insecticidal knockdown (‘fogging’). This resulted in an exhaustive collection of > 46,000
specimens from 24 major taxonomic/functional groups. Abundance distributions were
markedly negative exponential, indicating high abundance variability in tree crowns.
Individuals of six pertinent orders were identified to species level, returning > 3,100
individuals from 175 species and 52 families. This high diversity did marginally differ
across habitats, with slightly higher species richness in edge canopies. However,
communities in edge crowns were noticeably more heterogeneous than those in the
forest interior, possibly due to higher variability in environmental edge conditions. In
total, 49 species with protective value were identified, of which only one showed
habitat preferences (for near-natural interior forests). Among them, six species (all
beetles, Coleoptera) were classified as ‘priority species’ for conservation efforts. Hence,
beech canopies of the Northern Palatinate highlands can be considered strongholds of
insect biodiversity, incorporating many species of particular protective value.
The intricacy of plant-herbivore interaction networks and their relationship to
forest fragmentation is largely unexplored, particularly in Central Europe. Illumination
of this matter is all the more important, as ecological networks are highly relevant for
ecosystem stability, particularly in the face of additional anthropogenic disturbances,
such as climate change. Hence, plant-herbivore interaction networks (PHNs) were
constructed from woody plants and their associated herbivores, sampled alive in the
understory. Herbivory verification was achieved using no-choice-feeding assays, as well
as literature references. In total, networks across small forest fragments, edges, and
the forest interior consisted of 696 interactions. Network complexity and trophic niche
redundancy were compared across habitats using a rarefaction-like resampling
procedure. PHNs in fragmentation affected forest habitats were significantly more
complex, as well as more redundant in their realized niches, despite being composed of
relatively more specialist species. Furthermore, network robustness to climate change
was quantified utilizing four different scenarios for climate change susceptibility of
involved plants. In this procedure, remaining herbivores in the network were measured
upon successive loss of their host plant species. Consistently, PHNs in edges (and to a
smaller degree in small fragments) withstood primary extinction of plant species
longer, making them more robust. This was attributed to the high prevalence of
heat/drought-adapted species, as well as to beneficial effects of network topography
(complexity and redundancy). Consequently, strong correlative relationships were
found between realized niche redundancy and climate change robustness of PHNs.
This was both the first time that biologically realistic extinctions (instead of e.g.random extinctions) were used to measure network robustness, and that topographical
network parameters were identified as potential indicators for network robustness
against climate change.
In synthesis, in the light of global biotic degradation due to human forest
modification, the necessity to differentiate must be claimed. Ecosystems react
differently to anthropogenic disturbances, and it seems the particular features present
in Central European forests (ancient deforestation, extensive management, and, most
importantly, high richness in open-forest plant species) cause partly opposed patterns
to other biomes. Lenient microclimates and diverse plant communities facilitate
equally diverse herbivore assemblages, and hence complex and robust networks,
opposed to the forest interior. Therefore, in the reality of extensively used cultural
landscapes, fragmentation affected forest ecosystems, particularly forest edges, can be
perceived as reservoir for biodiversity, and ecosystem functionality. Nevertheless, as
practically all forest habitats considered in this thesis are under human cultivation,
recommendations for ecological enhancement of all forest habitats are discussed.
Membrane proteins are generally soluble only in the presence of detergent micelles or other membrane-mimetic systems, which renders the determination of the protein’s molar mass or oligomeric state difficult. Moreover, the amount of bound detergent varies drastically among different proteins and detergents. However, the type of detergent and its concentration have a great influence on the protein’s structure, stability, and functionality and the success of structural and functional investigations and crystallographic trials. Size-exclusion chromatography, which is commonly used to determine the molar mass of water-soluble proteins, is not suitable for detergent-solubilised proteins because
the protein–detergent complex has a different conformation and, thus, commonly exhibits
a different migration behaviour than globular standard proteins. Thus, calibration curves obtained with standard proteins are not useful for membrane-protein analysis. However,
the combination of size-exclusion chromatography with ultraviolet absorbance, static light scattering, and refractive index detection provides a tool to determine the molar mass of protein–detergent complexes in an absolute manner and allows for distinguishing the contributions of detergent and protein to the complex.
The goal of this thesis was to refine the standard triple-detection size-exclusion chromatography measurement and data analysis procedure for challenging membrane-protein samples, non-standard detergents, and difficult solvents such as concentrated denaturant solutions that were thought to elude routine approaches. To this end, the influence of urea on the performance of the method beyond direct influences on detergents and proteins was investigated with the help of the water-soluble bovine serum albumin. On the basis of
the obtained results, measurement and data analysis procedures were refined for different detergents and protein–detergent complexes comprising the membrane proteins OmpLA and Mistic from Escherichia coli and Bacillus subtilis, respectively.
The investigations on mass and shape of different detergent micelles and the compositions of protein–detergent complexes in aqueous buffer and concentrated urea solutions
showed that triple-detection size-exclusion chromatography provides valuable information
about micelle masses and shapes under various conditions. Moreover, it is perfectly suited for the straightforward analysis of detergent-suspended proteins in terms of composition and oligomeric state not only under native but, more importantly, also under denaturing conditions.
Cells and organelles are enclosed by membranes that consist of a lipid bilayer harboring highly
diverse membrane proteins (MPs). These carry out vital functions, and α-helical MPs, in
particular, are of outstanding pharmacological importance, as they comprise more than half of
all drug targets. However, knowledge from MP research is limited, as MPs require membranemimetic
environments to retain their native structures and functions and, thus, are not readily
amenable to in vitro studies. To gain insight into vectorial functions, as in the case of channels
and transporters, and into topology, which describes MP conformation and orientation in the
context of a membrane, purified MPs need to be reconstituted, that is, transferred from detergent
micelles into a lipid-bilayer system.
The ultimate goal of this thesis was to elucidate the membrane topology of Mistic, which is
an essential regulator of biofilm formation in Bacillus subtilis consisting of four α-helices. The
conformational stability of Mistic has been shown to depend on the presence of a hydrophobic
environment. However, Mistic is characterized by an uncommonly hydrophilic surface, and
its helices are significantly shorter than transmembrane helices of canonical integral MPs.
Therefore, the means by which its association with the hydrophobic interior of a lipid bilayer
is accomplished is a subject of much debate. To tackle this issue, Mistic was produced and
purified, reconstituted, and subjected to topological studies.
Reconstitution of Mistic in the presence of lipids was performed by lowering the detergent
concentration to subsolubilizing concentrations via addition of cyclodextrin. To fully exploit
the advantages offered by cyclodextrin-mediated detergent removal, a quantitative model was
established that describes the supramolecular state of the reconstitution mixture and allows
for the prediction of reconstitution trajectories and their cross points with phase boundaries.
Automated titrations enabled spectroscopic monitoring of Mistic reconstitutions in real time.
On the basis of the established reconstitution protocol, the membrane topology of Mistic was
investigated with the aid of fluorescence quenching experiments and oriented circular dichroism
spectroscopy. The results of these experiments reveal that Mistic appears to be an exception
from the commonly observed transmembrane orientation of α-helical MPs, since it exhibits
a highly unusual in-plane topology, which goes in line with recent coarse-grained molecular
dynamics simulations.