Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

A Parallel Coupled Lattice Boltzmann-Volume of Fluid Framework for Modeling Porous Media Evolution

In this paper, we present a framework for the modeling and simulation of a subset of physical/chemical processes occurring on different spatial and temporal scales in porous materials. In order to improve our understanding of such processes on multiple spatio-temporal scales, small-scale simulations of transport and reaction are of vital importance. Due to the geometric complexity of the pore space and the need to consider a representative elementary volume, such simulations require substantial numerical resolutions, leading to potentially huge computation times. An efficient parallelization of such numerical methods is thus vital to obtain results in acceptable wall-clock time. The goal of this paper was to improve available approaches based on lattice Boltzmann methods (LBMs) to reliably and accurately predict the combined effects of mass transport and reaction in porous media. To this end, we relied on the factorized central moment LBM as a second-order accurate approach for modeling transport. In order to include morphological changes due to the dissolution of the solid phase, the volume of fluid method with the piece-wise linear interface construction algorithm was employed. These developments are being integrated into the LBM research code VirtualFluids. After the validation of the analytic test cases, we present an application of diffusion-controlled dissolution for a pore space obtained from computer tomography (CT) scans

Exceptionally sweet - Studies on the bacterial arginine rhamnosyltransferase EarP

Bacterial protein glycosylation affects numerous cellular properties, including physiology and pathogenicity. The transfer of carbohydrates to a nitrogen atom is known as N glycosylation and almost exclusively occurs on asparagine side chains. In contrast, EarP represents a novel type of arginine-modifying glycosyltransferases. This enzyme uses TDP β-L-rhamnose as a donor substrate to activate the specialized translation elongation factor P (EF-P) in about 10 % of sequenced bacteria, including the clinically relevant species Pseudomonas aeruginosa and Neisseria meningitidis. The post-translational modification of EF-P is crucial for bacterial fitness and also constitutes a prerequisite for virulence. As the amido group of asparagine and the arginine guanidinium are chemically distinct, the activation of the latter might be based on a so far unsolved molecular mechanism. Consequently, the structural characterization of EarP and its products is of clinical and functional importance. In this regard, NMR analyses unambiguously identified the product of the glycosylation reaction as α-rhamnosyl-arginine. Thus, EarP inverts the anomeric configuration of rhamnose during the reaction. Anomer-specific mono-rhamnosyl-arginine-containing peptides were synthetized and used to raise antibodies against the modified side chain. These immunoglobulins were characterized with respect to their sensitivity and specificity towards the target epitope and used to determine enzyme kinetics of EarP. X-ray crystallography identified EarP as a member of the inverting GT-B superfamily and revealed the site for donor binding. Bioinformatic and mutant analyses elucidated the functional significance of several amino acids in orienting the nucleotide sugar and demonstrated the importance of two highly conserved aspartates for catalysis. Additionally, NMR titration experiments revealed that EarP mainly binds the N-terminal β barrel domain of its acceptor substrate EF-P. This information was utilized to generate the first synthetic target for EarP-mediated protein modification. The structurally but not sequentially related EF-P homologue from E. coli is naturally activated by lysylation of a lysine side chain. Successive mutation not only allowed modification but also activation of E. coli EF-P by the non-cognate and EarP-mediated rhamnosylation. This thesis provides new insights into the structure-function relationship of inverting arginine glycosylation. Additionally, it lays the groundwork for the application of EarP in synthetic biology and clinical research.Die Glykosylierung bakterieller Proteine beeinflusst zahlreiche zelluläre Eigenschaften wie Physiologie und Pathogenität. Die Übertragung von Kohlenhydraten auf ein Stickstoffatom wird als N-Glykosylierung bezeichnet und erfolgt fast ausschließlich an Asparagin-Seitenketten. Im Gegensatz dazu gehört EarP zu einer neuen Klasse von Arginin-modifizierenden Glykosyltransferasen. In etwa 10 % der sequenzierten Bakterien, einschließlich der klinisch relevanten Spezies Pseudomonas aeruginosa und Neisseria meningitidis, verwendet dieses Enzym TDP-β-L-rhamnose als Donorsubstrat zur Aktivierung des spezialisierten Translationselongationsfaktors P (EF-P). Die post-translationale Modifikation von EF-P ist von entscheidender Bedeutung für die bakterielle Fitness und eine Voraussetzung für Virulenz. Da die Amidogruppe von Asparagin und die Guanidinogruppe von Arginin chemisch unterschiedlich sind, erfolgt die Aktivierung der letzteren durch einen bisher unerforschten molekularen Mechanismus. Folglich ist die strukturelle Charakterisierung von EarP und seinen Katalyseprodukten sowohl von medizinischer als auch funktioneller Bedeutung. Mittels NMR wurde zunächst das Produkt der Glykosylierungsreaktion von EarP eindeutig als α-Rhamnosyl-Arginin identifiziert. Somit invertiert EarP die anomere Konfiguration von Rhamnose während der Reaktion. Anomer-spezifische mono-Rhamnosyl-Arginin enthaltende Peptide wurden synthetisiert und zur Generierung von Antikörpern verwendet. Diese Immunglobuline wurden hinsichtlich Sensitivität und Spezifität gegenüber dem Epitop charakterisiert und zur Bestimmung der Enzymkinetik von EarP verwendet. Die Kristallstrukturanalyse von EarP ermöglichte nicht nur eine Zuordnung des Enzyms zur Superfamilie der invertierenden GT-B-Glykosyltransferasen, sondern zeigte auch die Position der Donorbindestelle auf. Weitere bioinformatische und Mutagenese-basierte Studien führten zur Identifizierung von zwei für die Katalyse wichtigen Aspartaten sowie von mehreren Aminosäuren, die für die Orientierung des Nukleotidzuckers von Bedeutung sind. NMR-Titrationen ergaben, dass EarP hauptsächlich die N-terminale β-Barreldomäne des Akzeptorsubstrates EF-P bindet. Diese Information wurde verwendet, um den ersten synthetischen Akzeptor für eine EarP-vermittelte Proteinmodifikation zu generieren. Das strukturell, aber nicht sequentiell verwandte EF-P-Homolog von E. coli wird natürlicherweise durch Lysylierung einer Lysin-Seitenkette aktiviert. Infolge sukzessiver Aminsoäureaustausche wurde nicht nur die Modifikation von E. coli EF-P durch eine EarP vermittelte Rhamnosylierung erreicht, sondern auch die Aktivierung dieses Elongationsfaktors. Diese Arbeit liefert somit neue Erkenntnisse über die Struktur-Funktionsbeziehung der invertierenden Arginin-Glykosylierung. Darüber hinaus legt sie den Grundstein für die Anwendung von EarP in der Synthetischen Biologie und der klinischen Forschung

Test-retest reliability of visual-evoked potential habituation

Objective Habituation of visual-evoked potentials (VEPs) is typically described as deficient interictally in migraine patients, supposedly indicating altered cortical excitability. Use of this parameter for monitoring changes over time, e.g. under treatment, requires demonstration of test-retest reliability. Methods VEPs were recorded interictally in 41 episodic migraine patients and 40 controls. N75-P100 amplitudes were measured over six consecutive blocks of 75 VEPs each. Amplitude regression slopes and block ratios were used to quantify VEP habituation. Test-retest reliability was assessed over 15 minutes and two to three weeks. Results Controls showed significantly more negative VEP habituation slopes than migraine patients (-0.210.40 vs. 0.04 +/- 0.46 mu V/block, p<0.05). Results were similar for block ratios, though, in the migraine group, VEP habituation significantly increased from test to two- to three-week retest (p<0.05). In addition, VEP habituation test-retest correlations were mostly poor both in migraine patients and controls (intraclass correlation coefficients, 15 minutes: -0.13 to 0.30, two to three weeks: 0.07 to 0.59). Conclusions Deficient VEP habituation in migraine was confirmed. However, the test-retest reliability of VEP habituation was rather weak. Therefore, we suggest that VEP habituation should be used for evaluation of cortical excitability under treatment only at the group level and only when a control group with sham treatment is included

Test-retest reliability of visual-evoked potential habituation

Objective Habituation of visual-evoked potentials (VEPs) is typically described as deficient interictally in migraine patients, supposedly indicating altered cortical excitability. Use of this parameter for monitoring changes over time, e.g. under treatment, requires demonstration of test-retest reliability. Methods VEPs were recorded interictally in 41 episodic migraine patients and 40 controls. N75-P100 amplitudes were measured over six consecutive blocks of 75 VEPs each. Amplitude regression slopes and block ratios were used to quantify VEP habituation. Test-retest reliability was assessed over 15 minutes and two to three weeks. Results Controls showed significantly more negative VEP habituation slopes than migraine patients (-0.210.40 vs. 0.04 +/- 0.46 mu V/block, p<0.05). Results were similar for block ratios, though, in the migraine group, VEP habituation significantly increased from test to two- to three-week retest (p<0.05). In addition, VEP habituation test-retest correlations were mostly poor both in migraine patients and controls (intraclass correlation coefficients, 15 minutes: -0.13 to 0.30, two to three weeks: 0.07 to 0.59). Conclusions Deficient VEP habituation in migraine was confirmed. However, the test-retest reliability of VEP habituation was rather weak. Therefore, we suggest that VEP habituation should be used for evaluation of cortical excitability under treatment only at the group level and only when a control group with sham treatment is included

Mesoscopic Methods in Engineering and Science

(First paragraph) Matter, conceptually classified into fluids and solids, can be completely described by the microscopic physics of its constituent atoms or molecules. However, for most engineering applications a macroscopic or continuum description has usually been sufficient, because of the large disparity between the spatial and temporal scales relevant to these applications and the scales of the underlying molecular dynamics. In this case, the microscopic physics merely determines material properties such as the viscosity of a fluid or the elastic constants of a solid. These material properties cannot be derived within the macroscopic framework, but the qualitative nature of the macroscopic dynamics is usually insensitive to the details of the underlying microscopic interactions