Protein PCS NMR spectroscopy under physiological conditions: development of a new high yield cyclization method for rigidified DOTA-based tags with sulfhydryl-reactive activators forming a reductively stable linkage

This thesis focuses on the development of a new synthetic strategy towards twelve-membered tetraaza macrocycles and the synthesis of new sulfhydryl reactive linker moieties for the application in pseudocontact shift (PCS) NMR spectroscopy. The first part addresses the development of a new robust synthetic procedure for the selective synthesis of twelve-membered tetraaza macrocycles using natural amino acids and derivatives as cheap chiral building blocks. Cyclization of linear tetrapeptides was attempted but due to the preferred trans configuration of the peptide bond only small amounts of the cyclic tetrapeptide were obtained. Tetraalanine was reduced to tetraalaninol and the cyclization of tetraalaninol in solution was attempted using a preorganized bisaminal approach. This approach was unsuccessful and no product could be isolated. A more flexible mixed amine / amide tetramer was synthesized and successfully cyclized under modest dilution conditions forming a C2 symmetric twelve-membered bislactam. This reaction was highly efficient and afforded various bislactams based on alanine, alanine / valine and alanine / serine in good yields. From these bislactams various valuable intermediates for further tag synthesis are accessible as well as the final tetraaza macrocycles M4-cyclen, M2P2-cyclen and M3O1-cyclen. The newly developed approach allows the synthesis of a variety of differently substituted twelvemembered macrocycles. The second part primarily focuses on the development of new sulfhydrylreactive linker moieties for site-selective protein tagging. The widely used formation of a disulphide linkage has the inherent problem of being unstable under reductive conditions as they are found in living cells, limiting the range of application to buffered in-vitro applications. Pyridine phenyl sulfone based tags were synthesized and in-cell NMR experiments were conducted with the B1 domain of the streptococcal protein G (GB1). The structure of the protein was successfully calculated with the Rosetta approach using only pseudocontact shifts and residual dipolar couplings (RDCs) obtained from in-cell experiments in oocytes. We proved for the first time that accurate structures of proteins inside cells can be generated solely from experimental PCSs and RDCs within the Rosetta package. The conjugation speed was dramatically increased for the fluorine substituted pyridine phenyl sulfone analogue decreasing the reaction time from 24 h at 40 °C to six hours at 20-25 °C. Further enhancement was achieved using a pyridine thiazol allowing efficient tagging in less than one hour at 20-25 °C and pH 7.0. The last part deals with various different applications of M8-DOTA in order to solve structural or biological problems. Gd-M8-DOTA was selectively coupled to the Pittsburgh compound B (PiB) for in-vivo magnetic resonance imaging experiments. PiB strongly interacts with the amyloid plaques found in Alzheimer’s disease and thus PiB-Gd-M8-DOTA allows detecting amyloid plaques by MRI. A second collaboration project addresses the problems found in the assignment of a hexa-polyproline. Due to the repetitive nature of this peptide the chemical shift dispersion was small. Increasing the chemical shift range found in hexa-polyproline was the goal of the attempted conjugation of M8-DOTA to the hexa-polyproline. We showed that M8-DOTA can not be coupled to the N-terminal aniline moiety and a less sterically demanding amine linker is required. Prospective experiments were carried out on Eu-M8-DOTA and its closely related derivatives, Eu-M4-DOTA and Eu-DOTA, in order to check for possible applications in Förster resonance energy transfer (FRET) spectroscopy. All complexes were not luminescence under UV irradiation and a UV harvesting molecule was required. Eu-azaxanthon-M7-DOTA showed excellent luminescence properties required for potential applications in FRET spectroscopy. Preliminary experiments with Tb-M7PyThiazol-DOTA showed similar properties with a red shifted UV absorption maximum (from 250 nm to 300 nm) indicating great potential for FRET spectroscopy

Entrained Macrocryst Minerals as a Key to the Source Region of Olivine Nephelinites: Humberg, Kaiserstuhl, Germany

Olivine nephelinites commonly contain macrocrysts of olivine and clinopyroxene. Some of these macrocrysts might represent fragments of the source region of the host magma transported to the Earth's surface. If this hypothesis is correct these fragments can be used to characterize the composition of the source region and to put constraints on the magma generation process. In this study, we investigate the origin of macrocrysts and mineral aggregates from an olivine nephelinite from the Kaiserstuhl, Germany. We focus on clinopyroxenes (Cpx), which can be divided into three groups. Cpx I is relict Cpx from aggregates with deformed olivine that is depleted in Ca and characterized by strong light rare earth element (LREE) fractionation, low Ti/Eu and negative high field strength element (HFSE) anomalies. Its geochemical signature is consistent with formation by carbonatite metasomatism and with equilibration in the presence of orthopyroxene. Cpx II is Ca-rich Cpx, forming both aggregates with deformed olivine and individual macrocrysts. The LREE, as for Cpx I, are strongly fractionated. Convex REE patterns may be present. The depletion in HFSE is less pronounced. Cpx III is oscillatory zoned Cpx phenocrysts showing enrichment in Ca, convex REE patterns and no HFSE anomalies. The transition in the trace element abundances between the Cpx of the three groups is gradual. However, Cpx I and II did not crystallize from the host magma, as demonstrated by the presence of kink-bands and undulose extinction in the associated olivine and by the composition of alkali aluminosilicate glass inclusions in Cpx II. Based on the Cpx relationships, we interpret the studied suite of macrocrysts and mineral aggregates as a mixture of disintegrated fragments of the source region of the host olivine nephelinite. The process of melt generation was multi-stage. A primary carbonatite melt ascending from deeper levels in the mantle, probably from the dolomite-garnet peridotite stability field, reacted with mantle peridotite along the solidus ledge in the system lherzolite-CO2 (< 20-22 kbar) and started to crystallize carbonate minerals. Because of its low solidus temperature, the resulting carbonate-wehrlite assemblage melted incongruently with the formation of additional clinopyroxene. The carbonatite melt evolved during crystallization of carbonate minerals and concomitant incongruent melting of the carbonate-wehrlite, accompanied by the segregation of incipient alkali aluminosilicate melts. As a consequence of fast reaction rates in the presence of a carbonatite melt, this process probably took place under disequilibrium conditions. Further melting of the assemblage wehrlite + alkali aluminosilicate melt led to the generation of the olivine nephelinite magma. It entrained fragments of the wehrlite and brought them to the surfac

Plagioclase Peridotites in Ocean-Continent Transitions: Refertilized Mantle Domains Generated by Melt Stagnation in the Shallow Mantle Lithosphere

The existence of different mantle domains exposed in ocean-continent transition zones provides a framework for understanding the generation of ultramafic seafloor along magma-poor rifted margins. In this study we present detailed petrological and geochemical data on peridotites from the Eastern Central Alps ophiolites in Switzerland and Italy to identify different mantle domains, to estimate the extent of refertilization, and to test whether refertilization is associated with a thermal signature that has important implications for geophysical interpretations of ocean-continent transitions. The compositions of clinopyroxene, orthopyroxene and spinel clearly reflect the different mantle domains. Relative to clinopyroxenes from spinel peridotites, clinopyroxenes from plagioclase peridotites have lower Na2O and Sr contents, but higher middle to heavy rare earth element ratios and Zr concentrations, and different Sc-V relationships. Spinels in plagioclase peridotites have higher TiO2 and lower Mg-numbers compared with those in spinel peridotites. Mineral-mineral trace element partitioning suggests that spinel peridotites equilibrated at substantially lower temperatures than plagioclase peridotites. The temperature difference between the spinel and plagioclase peridotites indicates an important thermal boundary between the two. The geochemical data show systematic spatial variations. A heterogeneous, ‘subcontinental domain' with no syn-rift melt imprint is separated from a ‘refertilized domain' that exhibits a complex history of regional-scale melt infiltration and melt-rock reaction, which has erased most of the ancient history. Simple calculations suggest that up to 12% of mid-ocean ridge basalt-type melt can be stored in plagioclase peridotite, relative to a depleted residue. Such a ‘lithospheric sponge' provides an explanation for the fertile compositions of the peridotites and the rare occurrence of volcanic rocks in magma-poor rifted margins. We suggest that magma-poor vs magma-rich margins are largely determined by the efficiency of melt extraction and not so much by melt generation processes, given a similar initial composition of the upwelling mantle. It is proposed that refertilization increases textural diversity and chemical heterogeneity related to shallow crystallization in the mantle lithospher

Agreement of benchmarking high antimicrobial usage farms based on either animal treatment index or number of national defined daily doses

Introduction: While treatment frequency as an indicator of antimicrobial consumption is often assessed using defined doses, it can also be calculated directly as an Animal Treatment Index (ATI). In this study, the correlation of calculating antimicrobial usage on Swiss pig farms using either national Defined Daily Doses (DDDch) or an ATI (number of treatments per animal per year) and the agreement between the different methods for the identification of high usage farms were investigated. Material and Methods: The antimicrobial consumption of 893 Swiss pig herds was calculated separately for suckling piglets, weaned piglets, fattening pigs, lactating and gestating sows using the indicators nDDDch (number of DDDch) per animal per year and ATI. Correlations between the indicators were investigated by calculating Spearman's Rho coefficients. The 5, 10, and 25% highest usage farms were determined by applying both methods and the interrater reliability was described using Cohen's Kappa coefficients and visualized by Bland-Altman plots. Results: The Spearman's Rho coefficients showed strong correlations (r > 0.5) between nDDDch/animal/year and ATI. The lowest coefficient was shown for the correlation of both indicators in gestating sows (r = 0.657) and the highest in weaned piglets (r = 0.910). Kappa coefficients identifying high usage farms were the highest in weaned piglets (k = 0.71, 0.85, and 0.91, respectively for 5, 10, and 25% most frequent users) and the lowest in gestating sows (k = 0.54, 0.58, and 0.55 for 5, 10, and 25% most frequent users). Conclusions: In general, the investigated indicators showed strong correlations and a broad agreement in terms of the calculated levels of antimicrobial usage and the identification of high usage farms. Nevertheless, a certain proportion of the farms were defined differently depending on the indicator used. These differences varied by age category and were larger in all age categories except weaned piglets when a higher percentage benchmark was used to define high usage farms. These aspects should be considered when designing scientific studies or monitoring systems and considering which indicator to use

In-Cell Protein Structures from 2D NMR Experiments

In-cell NMR spectroscopy provides atomic resolution insights into the structural properties of proteins in cells, but it is rarely used to solve entire protein structures de novo. Here, we introduce a paramagnetic lanthanide-tag to simultaneously measure protein pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) to be used as input for structure calculation routines within the Rosetta program. We employ this approach to determine the structure of the protein G B1 domain (GB1) in intact Xenopus laevis oocytes from a single set of 2D in-cell NMR experiments. Specifically, we derive well-defined GB1 ensembles from low concentration in-cell NMR samples (∼50 μM) measured at moderate magnetic field strengths (600 MHz), thus offering an easily accessible alternative for determining intracellular protein structures

Bicyclic Phenyl–Ethynyl Architectures: Synthesis of a 1,4-Bis(phenylbuta-1,3-diyn-1-yl) Benzene Banister

The novel diacetylene bridged terphenylic macrocycle 1 is presented and discussed in the context of rotationally restricted “Geländer” oligomers. The 1,4‐bis(phenylbuta‐1,3‐diyn‐1‐yl) benzene bridge of diacetylene 1 is significantly longer than its terphenyl backbone, forcing the bridge to bend around the central pylon. The synthesis of molecule 1 is based to a large extent on acetylene scaffolding strategies, profiting from orthogonal alkyne protection groups to close both macrocyclic subunits by oxidative acetylene coupling sequentially. The spatial arrangement and the dynamic enantiomerization process of the bicyclic target structure 1 are analyzed. In‐depth NMR investigations not only reveal an unexpected spatial arrangement with both oligomer strands bent alongside the backbone, but also display the limited stability of the model compound in the presence of molecular oxygen

Bicyclic Phenyl-Ethynyl Architectures: Synthesis of a 1,4-Bis(phenylbuta-1,3-diyn-1-yl) Benzene Banister

The novel diacetylene bridged terphenylic macrocycle 1 is presented and discussed in the context of rotationally restricted "Gelander" oligomers. The 1,4-bis(phenylbuta-1,3-diyn-1-yl) benzene bridge of diacetylene 1 is significantly longer than its terphenyl backbone, forcing the bridge to bend around the central pylon. The synthesis of molecule 1 is based to a large extent on acetylene scaffolding strategies, profiting from orthogonal alkyne protection groups to close both macrocyclic subunits by oxidative acetylene coupling sequentially. The spatial arrangement and the dynamic enantiomerization process of the bicyclic target structure 1 are analyzed. In-depth NMR investigations not only reveal an unexpected spatial arrangement with both oligomer strands bent alongside the backbone, but also display the limited stability of the model compound in the presence of molecular oxygen

Petrology and Mineral Chemistry of Lower Crustal Intrusions: the Chilas Complex, Kohistan (NW Pakistan)

Mineral major and trace element data are presented for the main rock units of the Chilas Complex, a series of lower crustal intrusions emplaced during initial rifting within the Mesozoic Kohistan (paleo)-island arc (NW Pakistan). Detailed field observations and petrological analysis, together with geochemical data, indicate that the two principal units, ultramafic rocks and gabbronorite sequences, originate from a common parental magma, but evolved along different mineral fractionation trends. Phase petrology and mineral trace element data indicate that the fractionation sequence of the ultramafic rocks is dominated by the crystallization of olivine and clinopyroxene prior to plagioclase, whereas plagioclase precedes clinopyroxene in the gabbronorites. Clinopyroxene in the ultramafic rocks (with Mg-number [Mg/(Fetot + Mg] up to 0·95) displays increasing Al2O3 with decreasing Mg-number. The light rare earth element depleted trace element pattern (CeN/GdN ∼0·5-0·3) of primitive clinopyroxenes displays no Eu anomaly. In contrast, clinopyroxenes from the gabbronorites contain plagioclase inclusions, and the trace element pattern shows pronounced negative anomalies for Sr, Pb and Eu. Trace element modeling indicates that in situ crystallization may account for major and trace element variations in the gabbronorite sequence, whereas the olivine-dominated ultramafic rocks show covariations between olivine Mg-number and Ni and Mn contents, pointing to the importance of crystal fractionation during their formation. A modeled parental liquid for the Chilas Complex is explained in terms of mantle- and slab-derived components, where the latter component accounts for 99% of the highly incompatible elements and between 30 and 80% of the middle rare earth elements. The geochemical characteristics of this component are similar to those of a low percentage melt or supercritical liquid derived from subducted mafic crust. However, elevated Pb/Ce ratios are best explained by additional involvement of hydrous fluids. In accordance with the crystallization sequence, the subsolidus metamorphic reactions indicate pressures of 0·5-0·7 GPa. Our data support a model of combined flux and decompression melting in the back-ar

New Lanthanide Chelating Tags for PCS NMR Spectroscopy with Reduction Stable, Rigid Linkers for Fast and Irreversible Conjugation to Proteins

Lanthanide chelating tags (LCTs) have been used with great success for determining structures and interactions of proteins and other biological macromolecules. Recently LCTs have also been used for in-cell NMR spectroscopy, but the bottleneck especially for demanding applications like pseudocontact shift (PCS) NMR is the sparse availability of suitable tags that allow for site-selective, rigid, irreversible, fast, and quantitative conjugation of chelated paramagnetic lanthanide ions to proteins via reduction stable bonds. We report here several such tags and focus on a new pyridine thiazole derivate of DOTA, that combines high affinity, rigidity, and selectivity with unprecedented tagging properties. The conjugation to the cysteine thiol of the protein results in a reductively stable thioether bond and proceeds virtually quantitatively in less than 30 min at 100 μM protein concentration, ambient temperature, and neutral pH. Upon conjugation of the new tag to two single cysteine mutants of ubiquitin and a single cysteine mutant of human carbonic anhydrase type II (30 kDa) only one stereoisomer is formed (square antiprismatic coordination, Λ(δδδδ)) and large to very large pseudocontact shifts as well as large residual dipolar couplings (RDCs) are observed by NMR spectroscopy. The PCS and RDC show excellent agreement with the solid state structure of the proteins. We believe that the pyridine thiazole moiety reported here has the potential to serve as a thiole reactive group in various conjugation applications; furthermore, its terbium complex shows strong photoluminescence upon irradiation and may thus serve as a donor group for Förster resonance energy transfer spectroscopy

Unexpected dynamics in femtomolar complexes of binding proteins with peptides

Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders