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.

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.

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.

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.

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.

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.

Photoconversion mechanism of the second GAF domain of cyanobacteriochrome AnPixJ and the cofactor structure of its green-absorbing state.

Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C-D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.

Catalytic efficiency of dehaloperoxidase A is controlled by electrostatics--application of the vibrational Stark effect to understand enzyme kinetics.

The vibrational Stark effect is gaining popularity as a method for probing electric fields in proteins. In this work, we employ it to explain the effect of single charge mutations in dehaloperoxidase-hemoglobin A (DHP A) on the kinetics of the enzyme. In a previous communication published in this journal (BBRC 2012, 420, 733-737) it has been shown that an increase in the overall negative charge of DHP A through mutation causes a decrease in its catalytic efficiency. Here, by labeling the protein with 4-mercaptobenzonitrile (MBN), a Stark probe molecule, we provide further evidence that the diffusion control of the catalytic process arises from the electrostatic repulsion between the enzyme and the negatively charged substrate. The linear correlation observed between the nitrile stretching frequency of the protein-bound MBN and the catalytic efficiency of the single-site mutants of the enzyme indicates that electrostatic interactions play a dominant role in determining the catalytic efficiency of DHP A.

Effect of the protonation degree of a self-assembled monolayer on the immobilization dynamics of a [NiFe] hydrogenase.

Understanding the interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is important in the field of biotechnology and, in particular, for the development of biofuel cells. In this study, we investigated the adsorption behavior of the standard [NiFe] hydrogenase of Desulfovibrio gigas on amino-terminated alkanethiol self-assembled monolayers (SAMs) with different levels of protonation. Classical all-atom molecular dynamics (MD) simulations revealed a strong correlation between the adsorption behavior and the level of ionization of the chemically modified electrode surface. While the hydrogenase undergoes a weak but stable initial adsorption process on SAMs with a low degree of protonation, a stronger immobilization is observable on highly ionized SAMs, affecting protein reorientation and conformation. These results were validated by complementary surface-enhanced infrared absorption (SEIRA) measurements on the comparable [NiFe] standard hydrogenases from Desulfovibrio vulgaris Miyazaki F and allowed in this way for a detailed insight into the adsorption mechanism at the atomic level.

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.

Coupling of Ci-VSP modules requires a combination of structure and electrostatics within the linker.

The voltage-sensitive phosphatase Ci-VSP consists of an intracellular phosphatase domain (PD) coupled to a transmembrane voltage-sensor domain (VSD). Depolarization triggers the selective dephosphorylation of phosphoinositides. However, the molecular mechanisms of coupling are still elusive. To clarify the role of the VSD-PD linker as a putative partner for electrostatic interactions with the membrane, we carried out a cysteine-scanning mutagenesis of the whole motif M240-K257. Upon coexpression with PI(4,5)P(2)-sensitive KCNQ2/KCNQ3 channels in Xenopus oocytes, we identified four positions (A242C, R245C, K252C, and Y255C) with a completely abrogated PD activity. Because the mutation effect occurred periodically, we hypothesize that α-helical elements exist within the linker, with a gap near position S249. The combination of these results with the analysis of transient sensing currents of the VSD revealed distinct roles for the N-terminal (M240-S249) and C-terminal (Q250-K257) linker motifs in the VSD-PD coupling. According to our functional results, the computational structure prediction of the Q239-D258 fragment confirmed α-helical structures within the linker, with a short ß-turn around S249 in the activated conformation. Remarkably, the position K252 may be a candidate for interacting with the PD rather than for binding to the membrane. This provides the first insight (to our knowledge) into the direct intervention of the linker in the VSD-PD coupling process.

Adsorption of sulfite oxidase on self-assembled monolayers from molecular dynamics simulations.

Sulfite oxidase (SO) is an enzyme catalyzing the terminal step of the metabolism of sulfur-containing amino acids that is essential for almost all living organisms. The catalytic activity of SO in vertebrates strongly depends on the efficiency of the intramolecular electron transfer (IET) between the catalytic Moco domain and the cytochrome b5 (cyt b5) domain. The IET process is assumed to be mediated by large domain motions of the cyt b5 domains within the enzyme. Thus, the interaction of SO with charged surfaces may affect the mobility of the cyt b5 domain required for IET and consequently hinder SO activation. In this study, we present a molecular dynamics approach to investigating the ionic strength dependence of the initial surface adsorption of SO in two different conformations-the crystallographic structure and the model structure for an activated SO-onto mixed amino- and hydroxyl-terminated SAMs. The results show for both conformations at low ionic strengths a strong adsorption of the cyt b5 units onto the SAM, which inhibits the domain motion event required for IET. Under higher ion concentrations, however, the interaction with the surface is weakened by the negatively charged ions acting as a buffer and competing in adsorption with the cathodic cyt b5 domains. This competition prevents the immobilization of the cytochrome b5 units onto the surface, allowing the intramolecular domain motions favoring IET. Our predictions support the interpretation of recent experimental spectroelectrochemical studies on SO.

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.

Molecular dynamics simulations of the adsorption of bone morphogenetic protein-2 on surfaces with medical relevance.

Bone morphogenetic protein-2 (BMP-2) plays a crucial role in osteoblast differentiation and proliferation. Its effective therapeutic use for ectopic bone and cartilage regeneration depends, among other factors, on the interaction with the carrier at the implant site. In this study, we used classical molecular dynamics (MD) and a hybrid approach of steered molecular dynamics (SMD) combined with MD simulations to investigate the initial stages of the adsorption of BMP-2 when approaching two implant surfaces, hydrophobic graphite and hydrophilic titanium dioxide rutile. Surface adsorption was evaluated for six different orientations of the protein, two end-on and four side-on, in explicit water environment. On graphite, we observed a weak but stable adsorption. Depending on the initial orientation, hydrophobic patches as well as flexible loops of the protein were involved in the interaction with graphite. On the contrary, BMP-2 adsorbed only loosely to hydrophilic titanium dioxide. Despite a favorable interaction energy between protein and the TiO(2) surface, the rapid formation of a two-layer water structure prevented the direct interaction between protein and titanium dioxide. The first water adlayer had a strong repulsive effect on the protein, while the second attracted the protein toward the surface. For both surfaces, hydrophobic graphite and hydrophilic titanium dioxide, denaturation of BMP-2 induced by adsorption was not observed on the nanosecond time scale.

Insights into the structure of the active site of the O2-tolerant membrane bound [NiFe] hydrogenase of R. eutropha H16 by molecular modelling.

Structural models for the Ni-B state of the wild-type and C81S protein variant of the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha H16 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.

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 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.

Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach.

Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(-1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.