Diversity of rhodopsin cyclases in zoospore-forming fungi.

Light perception for orientation in zoospore-forming fungi is linked to homo- or heterodimeric rhodopsin-guanylyl cyclases (RGCs). Heterodimeric RGCs, first identified in the chytrid Rhizoclosmatium globosum, consist of an unusual near-infrared absorbing highly fluorescent sensitizer neorhodopsin (NeoR) that is paired with a visual light-absorbing rhodopsin responsible for enzyme activation. Here, we present a comprehensive analysis of the distribution of RGC genes in early-branching fungi using currently available genetic data. Among the characterized RGCs, we identified red-sensitive homodimeric RGC variants with maximal light activation close to 600 nm, which allow for red-light control of GTP to cGMP conversion in mammalian cells. Heterodimeric RGC complexes have evolved due to a single gene duplication within the branching of Chytridiales and show a spectral range for maximal light activation between 480 to 600 nm. In contrast, the spectral sensitivity of NeoRs is reaching into the near-infrared range with maximal absorption between 641 and 721 nm, setting the low energy spectral edge of rhodopsins so far. Based on natural NeoR variants and mutational studies, we reevaluated the role of the counterion-triad proposed to cause the extreme redshift. With the help of chimera constructs, we disclose that the cyclase domain is crucial for functioning as homo- or heterodimers, which enables the adaptation of the spectral sensitivity by modular exchange of the photosensor. The extreme spectral plasticity of retinal chromophores in native photoreceptors provides broad perspectives on the achievable spectral adaptation for rhodopsin-based molecular tools ranging from UVB into the near-infrared.

The opening dynamics of the lateral gate regulates the activity of rhomboid proteases.

Rhomboid proteases hydrolyze substrate helices within the lipid bilayer to release soluble domains from the membrane. Here, we investigate the mechanism of activity regulation for this unique but wide-spread protein family. In the model rhomboid GlpG, a lateral gate formed by transmembrane helices TM2 and TM5 was previously proposed to allow access of the hydrophobic substrate to the shielded hydrophilic active site. In our study, we modified the gate region and either immobilized the gate by introducing a maleimide-maleimide (M2M) crosslink or weakened the TM2/TM5 interaction network through mutations. We used solid-state nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, and molecular docking to investigate the resulting effects on structure and dynamics on the atomic level. We find that variants with increased dynamics at TM5 also exhibit enhanced activity, whereas introduction of a crosslink close to the active site strongly reduces activity. Our study therefore establishes a strong link between the opening dynamics of the lateral gate in rhomboid proteases and their enzymatic activity.

Potential Distribution across Model Membranes.

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercaptobenzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental-theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

A combination of solid-state NMR and MD simulations reveals the binding mode of a rhomboid protease inhibitor.

Intramembrane proteolysis plays a fundamental role in many biological and pathological processes. Intramembrane proteases thus represent promising pharmacological targets, but few selective inhibitors have been identified. This is in contrast to their soluble counterparts, which are inhibited by many common drugs, and is in part explained by the inherent difficulty to characterize the binding of drug-like molecules to membrane proteins at atomic resolution. Here, we investigated the binding of two different inhibitors to the bacterial rhomboid protease GlpG, an intramembrane protease characterized by a Ser-His catalytic dyad, using solid-state NMR spectroscopy. H/D exchange of deuterated GlpG can reveal the binding position while chemical shift perturbations additionally indicate the allosteric effects of ligand binding. Finally, we determined the exact binding mode of a rhomboid protease-inhibitor using a combination of solid-state NMR and molecular dynamics simulations. We believe this approach can be widely adopted to study the structure and binding of other poorly characterized membrane protein-ligand complexes in a native-like environment and under physiological conditions.

Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs.

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described - relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.

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

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.IMPORTANCE Fungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance.

Long-Range Modulations of Electric Fields in Proteins.

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ Eads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ Eint, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ Eads and Δ Eint, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ Eads) and attached to membranes (Δ Eads + Δ Eint).

Solvation of diclofenac in water from atomistic molecular dynamics simulations - interplay between solute-solute and solute-solvent interactions.

The solubility-permeability relationship of active pharmaceutical ingredients determines the efficacy of their usage. Diclofenac (DCL), which is a widely used nonsteroidal anti-inflammatory drug, is characterized by extremely good membrane permeability, but low water solubility limiting drug effectiveness. The present research focuses on the fundamental explanation of this limitation using the combination of ab initio and classical molecular dynamics simulations of different ionic forms of DCL in water, namely, ionized, un-ionized and the mixture of them both. The analysis of diclofenac solvation in an aqueous environment is used to understand the origin of drug precipitation, especially in gastric pH. The used computational approach reveals the formation of micelle-like self-associated aggregates of diclofenac in water as the result of intermolecular π-π interactions and C-Hπ hydrogen bonds. The DCL aggregation in water is shown to depend mostly on drug concentration, protonation and temperature of the aqueous environment. The detected self-association properties of the drug in water are likely to be of great importance during the development of new drug formulations and fabrication of drug adsorbents for wastewater.

Tracking the route of molecular oxygen in O2-tolerant membrane-bound [NiFe] hydrogenase.

[NiFe] hydrogenases catalyze the reversible splitting of H2 into protons and electrons at a deeply buried active site. The catalytic center can be accessed by gas molecules through a hydrophobic tunnel network. While most [NiFe] hydrogenases are inactivated by O2, a small subgroup, including the membrane-bound [NiFe] hydrogenase (MBH) of Ralstonia eutropha, is able to overcome aerobic inactivation by catalytic reduction of O2 to water. This O2 tolerance relies on a special [4Fe3S] cluster that is capable of releasing two electrons upon O2 attack. Here, the O2 accessibility of the MBH gas tunnel network has been probed experimentally using a "soak-and-freeze" derivatization method, accompanied by protein X-ray crystallography and computational studies. This combined approach revealed several sites of O2 molecules within a hydrophobic tunnel network leading, via two tunnel entrances, to the catalytic center of MBH. The corresponding site occupancies were related to the O2 concentrations used for MBH crystal derivatization. The examination of the O2-derivatized data furthermore uncovered two unexpected structural alterations at the [4Fe3S] cluster, which might be related to the O2 tolerance of the enzyme.

Structure, dynamics and kinetics of two-component Lantibiotic Lichenicidin.

Two variants of the two-component Lantibiotic Lichenicidin, produced by the strains B. Licheniformis VK21 and I89 (Lchα/ Lchß and Bliα/ Bliß peptides, respectively) have been investigated by means of 2 µs-long all-atom molecular dynamics simulations combined with Markov State Models. This rigorous statistical analysis enabled to evaluate the dynamic and kinetic properties of the aforementioned systems which are not accessible via experimental techniques. The structural flexibility characteristic of these small peptides is understood by a delicate equilibrium between random coil, α-helices and ß-sheet structures. The undergoing secondary structure transitions from an α-helix to a ß-sheet observed for Lchα and Lchß peptides, were not present in the Bliα component and provide new insights to understand their mechanism of action.

Switchable Redox Chemistry of the Hexameric Tyrosine-Coordinated Heme Protein.

Hexameric tyrosine-coordinated heme protein HTHP from Silicibacter pomeroyi has been shown to exhibit peroxidase- and catalase-like activity. In the present study, detailed spectroscopic and electrochemical investigations were performed to analyze the redox properties and active site structure of HTHP. Potentiometric titration of HTHP in solution revealed a single redox transition at -0.54 V (vs Ag/AgCl), indicating six structurally identical tyrosine coordinates hemes. Cyclic voltammetry (CV) of immobilized HTHP afforded a distinctly more positive redox potential (-0.17 V) but failed to detect a transition at -0.54 V. Conversely, surface enhanced RR (SERR) spectroscopy provided evidence for both high- and low-potential transitions and for a partial loss of heme in the reduced state. The high-potential CV-active redox transition is attributed to the hemes of the barrel-shaped HTHP in a wheel-like orientation on the surface. Supported by coarse-grained simulations and SERR spectroscopy, the majority of HTHP is concluded to adopt a reverse-disc orientation, accounting for the low-potential transition. In view of the striking similarity of HTHP to the heme carriers HasA or HmbR regarding redox potential, Fe-Tyr ligation, and heme release, we propose heme transport as an alternative or additional function.

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.

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.

The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions.

Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal-containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pKa of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.

Orientation-Controlled Electrocatalytic Efficiency of an Adsorbed Oxygen-Tolerant Hydrogenase.

Protein immobilization on electrodes is a key concept in exploiting enzymatic processes for bioelectronic devices. For optimum performance, an in-depth understanding of the enzyme-surface interactions is required. Here, we introduce an integral approach of experimental and theoretical methods that provides detailed insights into the adsorption of an oxygen-tolerant [NiFe] hydrogenase on a biocompatible gold electrode. Using atomic force microscopy, ellipsometry, surface-enhanced IR spectroscopy, and protein film voltammetry, we explore enzyme coverage, integrity, and activity, thereby probing both structure and catalytic H2 conversion of the enzyme. Electrocatalytic efficiencies can be correlated with the mode of protein adsorption on the electrode as estimated theoretically by molecular dynamics simulations. Our results reveal that pre-activation at low potentials results in increased current densities, which can be rationalized in terms of a potential-induced re-orientation of the immobilized enzyme.

Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\prime }}\hfill \atop {\rm app}\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.

Structural parameters controlling the fluorescence properties of phytochromes.

Phytochromes constitute a class of photoreceptors that can be photoconverted between two stable states. The tetrapyrrole chromophore absorbs in the red spectral region and displays fluorescence maxima above 700 nm, albeit with low quantum yields. Because this wavelength region is particularly advantageous for fluorescence-based deep tissue imaging, there is a strong interest to engineer phytochrome variants with increased fluorescence yields. Such targeted design efforts would substantially benefit from a deeper understanding of those structural parameters that control the photophysical properties of the protein-bound chromophore. Here we have employed resonance Raman (RR) spectroscopy and molecular dynamics simulations for elucidating the chromophore structural changes in a fluorescence-optimized mutant (iRFP) derived from the PAS-GAF domain of the bacteriophytochrome RpBphP2 from Rhodopseudomas palustris . Both methods consistently reveal the structural consequences of the amino acid substitutions in the vicinity of the biliverdin chromophore that may account for lowering the propability of nonradiative excited state decays. First, compared to the wild-type protein, the tilt angle of the terminal ring D with respect to ring C is increased in iRFP, accompanied by the loss of hydrogen bond interactions of the ring D carbonyl function and the reduction of the number of water molecules in that part of the chromophore pocket. Second, the overall flexibility of the chromophore is significantly reduced, particularly in the region of rings D and A, thereby reducing the conformational heterogeneity of the methine bridge between rings A and B and the ring A carbonyl group, as concluded from the RR spectra of the wild-type proteins.

The linker pivot in Ci-VSP: the key to unlock catalysis.

In the voltage-sensitive phosphatase Ci-VSP, conformational changes in the transmembrane voltage sensor domain (VSD) are transduced to the intracellular catalytic domain (CD) leading to its dephosphorylation activity against membrane-embedded phosphoinositides. The linker between both domains is proposed to be crucial for the VSD-CD coupling. With a combined approach of electrophysiological measurements on Xenopus oocytes and molecular dynamics simulations of a Ci-VSP model embedded in a lipid bilayer, we analyzed how conformational changes in the linker mediate the interaction between the CD and the activated VSD. In this way, we identified specific residues in the linker that interact with well-defined amino acids in one of the three loops forming the active site of the protein, named TI loop. With our results, we shed light into the early steps of the coupling process between the VSD and the CD, which are based on fine-tuned electrostatic and hydrophobic interactions between the linker, the membrane and the CD.