Insights into the structure of the active site of the O-2-tolerant membrane bound [NiFe] hydrogenase of R. eutropha H16 by molecular modelling

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Structural models for the Ni-B state of the wild-type and C81S protein variant of the membrane-bound [NiFe] hydrogenase from Ralstonia eutrophaH16 were derived by applying the homology model technique combined with molecular simulations and a hybrid quantum mechanical/molecular mechanical approach. The active site structure was assessed by comparing calculated and experimental IR spectra, confirming the view that the active site structure is very similar to those of anaerobic standard hydrogenases. In addition, the data suggest the presence of a water molecule in the second coordination sphere of the active centre.DFG, EXC 314, Unifying Concepts in Catalysi

Domain motions and electron transfer dynamics in 2Fe-superoxide reductase

Superoxide reductases are non-heme iron enzymes that represent valuable model systems for the reductive detoxification of reactive oxygen species. In the present study, we applied different theoretical methods to study the structural dynamics of a prototypical 2Fe-superoxide reductase and its influence on electron transfer towards the active site. Using normal mode and essential dynamics analyses, we could show that enzymes of this type are capable of well-defined, electrostatically triggered domain movements, which may allow conformational proofreading for cellular redox partners involved in intermolecular electron transfer. Moreover, these global modes of motion were found to enable access to molecular configurations with decreased tunnelling distances between the active site and the enzyme's second iron centre. Using all-atom classical molecular dynamics simulations and the tunnelling pathway model, however, we found that electron transfer between the two metal sites is not accelerated under these conditions. This unexpected finding suggests that the unperturbed enzymatic structure is optimized for intramolecular electron transfer, which provides an indirect indication of the biological relevance of such a mechanism. Consistently, efficient electron transfer was found to depend on a distinct route, which is accessible via the equilibrium geometry and characterized by a quasi conserved tyrosine that could enable multistep-tunnelling (hopping). Besides these explicit findings, the present study demonstrates the importance of considering both global and local protein dynamics, and a generalized approach for the functional analysis of these aspects is provided

Mapping local electric fields in proteins at biomimetic interfaces

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.We present a novel approach for determining the strength of the electric field experienced by proteins immobilised on membrane models. It is based on the vibrational Stark effect of a nitrile label introduced at different positions on engineered proteins and monitored by surface enhanced infrared absorption spectroscopy

A Computational Modeling Approach Predicts Interaction of the Antifungal Protein AFP from Aspergillus giganteus with Fungal Membranes via Its γ-Core Motif

This work is licensed under a Creative Commons Attribution 4.0 International License.Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.NIH GM31749NIH GM103426Deutsche Forschungsgemeinschaft (Cluster of Excellence ‘Unifying Concepts in Catalysis’ and SFB1078

The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on fluorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikDFG, 53182490, EXC 314: Unifying Concepts in Catalysi

Molekulardynamische Simulationen von Enzymen an Oberflächen

Die Immobilisierung von Biomolekülen auf verschiedenen Oberflächenmaterialien spielt eine bedeutende Rolle in einem weiten Forschungsfeld. Die Wechselwirkung zwischen großen Enzymen und organischen und inorganischen Materialien ist, unter anderem, entscheidend bei der Entwicklung von alternativen Energiequellen, der Biomedizin, der Biokatalyse, bei der Zelltransduktion und in der Grundlagenforschung. Ein Hauptproblem in diesem Zusammenhang ist das Fehlen von strukturellen und dynamischen Details auf atomarer Ebene. Das Schließen dieser Lücke in eine äußerst wichtige Herausforderung, da viele Prozesse, die für die korrekte Proteinfunktion und Oberflächennteraktion verantwortlich sind, immer noch schwer zu fassen und im Detail unbekannt sind. Leider ist die experimentelle Zugänglichkeit dieser Eigenschaften extrem schwierig und aufwendig. Aus diesem Grund sind theoretische Ansätze wie klassische Moleküldynamiksimulationen (MD Simulationen) zur Bestimmung dieser Kenngrößen von großem Nutzen. Klassische Moleküldynamik ist eine potentielle und etablierte Technik, um die Adsorption von Biomolekülen auf Oberflächenmaterialien zu beschreiben, und bietet zusätzlich zu diesen Einsichten in die Dynamik Informationen über die Proteinstabilität, die einen entscheidenden Punkt bei Oberflächenkontakt darstellt. In dieser Arbeit wurden die Interaktionsdynamiken von Cytochrom c, dem knochenbildenden Protein BMP-2, der Sulfitoxidase (SO), [NiFe] Hydrogenasen und der spannungsabhängigen Phosphatase von Ciona intestinalis (Ci-VSP) mit klassischen Moleküldynamiksimulationen untersucht. Trotz der weiten Streuung der Projekte und deren unterschiedlicher Motivation und Zielsetzung, stehen dennoch bei allen ungeklärte Protein-Oberflächeninteraktionen im Vordergrund. Die Simulationen zeigten, dass sogar kleine Variationen in einem Modell große Auswirkungen auf das Adsorptionsverhalten und die Konformation eines Biomoleküls an Oberflächen haben kann. Die hier untersuchten Änderungen umfassten Mutationen in den Enzymen, Modifikationen an den Oberflächen durch Beschichtung mit unterschiedlich funktionalisierten selbst-assemblierten Monoschichten (SAMs) und Unterschiede an Deskriptoren, die die Umgebungsbedingungen innerhalb des Modells beschreiben, wie beispielsweise der pH Wert oder die Ionenstärke. Hierdurch wurden strukturelle Reorganisationen innerhalb der Enzyme, die zu unterschiedlichen Interaktionen mit den Oberflächen führten, Reorientierungen der Adsorbaten bezüglich der Oberflächen und die Ausbildung von stabilen Wasser- oder Ionenschichten auf den Oberflächen beobachtet. Der Vergleich von diesen Ergebnissen der molekularen Modellierung mit experimentell bestimmten Daten von Kooperationspartnern zeigte eine gute Übereinstimmung und bot die Möglichkeit, Immobilisierungen an Oberflächen gerichtet zu verbessern und Proteinfunktionen detaillierter zu verstehen. Dennoch muss dieser vielversprechende Ansatz aus klassischen MD Simulationen in Kombination mit Experimenten vorsichtig behandelt werden, da sich die Zeitskalen der beiden Methoden, auf denen sie das System analysieren, stark von einander unterscheiden.The immobilization of biomolecules on various surface materials plays an important role in a wide field of research. The interaction of large enzymes with organic and inorganic devices is, inter alia, relevant in the sectors of alternative energies, biomedicine, biocatalysis, cell signalling, and basic research. One major drawback in this context is the lacking of structural and dynamic details on the atomic level. Closing this gap is a very important challenge because many processes accounting for the correct protein functionality or for the proper adsorbant-surface interaction are still elusive. Unfortunately, the experimental access and determination of these properties is extremely difficult and expensive. Therefore, theory, namely, classical all-atom molecular dynamics (MD) simulations, is needed to investigate these issues. Classical MD simulations are a potential and established technique to describe the initial adsorption of biomolecules onto surface materials on the atomic level and offer, additionally to these dynamics insights, information about the protein stability, which constitutes a crucial point upon surface contact. In this work, the surface interaction dynamics of cytochrome c, bone morphogenetic protein-2 (BMP-2), sulfite oxidase (SO), [NiFe] hydrogenases and Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) was investigated by classical all-atom MD simulations. The widely scattered projects are motivated by different goals and are settled in diverse fields of research, but all deal with protein-surface interactions that are not completely understood. The simulations demonstrated that even small changes in the model could strongly affect the adsorption and conformation of biomolecules on surfaces. The variations included mutations in the enzyme, modifications of the surface by coating it with differently functionalized self-assembled monolayers (SAMs) and alterations of environmental parameters, such as the pH value and ionic strength. In doing so, structural rearrangements within the enzyme leading to different interactions with the surface, reorientations of the adsorbate with respect to the surface and the formation of stable water or ion layers on the device competing with the biomolecule in adsorption were observed. Comparison between these findings of the molecular modelling approach and experimental data measured by cooperation partners showed a good agreement and offered a more precise way to improve surface immobilization and to understand protein functionality in more detail. Nevertheless, the potential of classical MD simulations combined with experimental work has to be treated carefully, because both methods analyse systems on different time scales

Three-Dimensional Structural Model of Chicken Liver Sulfite Oxidase in its Activated Form

Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate in almost all living organisms. In vertebrates, the catalytic process involves a rapid intramolecular electron transfer (IET) step between the molybdenum cofactor in the central domain and the heme in the cytochrome <i>b</i>5 domain. The large distance between redox centers observed in the crystal structure disagrees with the fast IET rates measured experimentally. This conflict was explained by postulating a major rearrangement of the cytochrome <i>b</i>5 domain toward the molybdopterin cofactor. Using steered molecular dynamics and molecular dynamics simulations, we generated a stable 3D structural model for chicken liver SO (CSO) in the activated form characterized by a short electron donor−acceptor distance consistent with the enzymes’ experimentally obtained electron transfer properties. IET rates for the active complex were estimated with the Pathway model. The good agreement between calculated and experimental IET rates supports our structural model for the active CSO

Three-Dimensional Structural Model of Chicken Liver Sulfite Oxidase in its Activated Form

Sulfite oxidase (SO) catalyzes the conversion of sulfite to sulfate in almost all living organisms. In vertebrates, the catalytic process involves a rapid intramolecular electron transfer (IET) step between the molybdenum cofactor in the central domain and the heme in the cytochrome <i>b</i>5 domain. The large distance between redox centers observed in the crystal structure disagrees with the fast IET rates measured experimentally. This conflict was explained by postulating a major rearrangement of the cytochrome <i>b</i>5 domain toward the molybdopterin cofactor. Using steered molecular dynamics and molecular dynamics simulations, we generated a stable 3D structural model for chicken liver SO (CSO) in the activated form characterized by a short electron donor−acceptor distance consistent with the enzymes’ experimentally obtained electron transfer properties. IET rates for the active complex were estimated with the Pathway model. The good agreement between calculated and experimental IET rates supports our structural model for the active CSO