Trifluoromethylated Proline Surrogates as Part of "Pro-Pro" Turn-Inducing Templates.

Proline is often found as a turn inducer in peptide or protein domains. Exploitation of its restricted conformational freedom led to the development of the d-Pro-l-Pro (corresponding to (R)-Pro-(S)-Pro) segment as a "templating" unit, frequently used in the design of ß-hairpin peptidomimetics, in which conformational stability is, however, inherently linked to the cis-trans isomerization of the prolyl amide bonds. In this context, the stereoelectronic properties of the CF3 group can aid in conformational control. Herein, the impact of α-trifluoromethylated proline analogues is examined for the design of enhanced ß-turn inducers. A theoretical conformational study permitted the dipeptide (R)-Pro-(R)-TfmOxa (TfmOxa: 2-trifluoromethyloxazolidine-2-carboxylic acid) to be selected as a template with an increased trans-cis rotational energy barrier. NMR spectroscopic analysis of the Ac-(R)-Pro-(R)-TfmOxa-(S)-Val-OtBu ß-turn model, obtained through an original synthetic pathway, validated the prevalence of a major trans-trans conformer and indicated the presence of an internal hydrogen bond. Altogether, it was shown that the (R)-Pro-(R)-TfmOxa template fulfilled all crucial ß-turn-inducer criteria.

Solvent and Autocatalytic Effects on the Stabilisation of the σ-Complex during Electrophilic Aromatic Chlorination.

The solvent and autocatalytic effects of the electrophilic aromatic chlorination of benzene are studied using a combined approach of static calculations and ab initio metadynamics simulations. Different possible reaction pathways are investigated and the influence of the solvents (CCl4 , acetonitrile and acetic acid) is thoroughly assessed. Our results show that the stability and lifetime of a charged σ-complex is increased by electrostatic stabilisation effects of the environment, which can originate from catalytic HCl, solvating effects of polar solvents (acetonitrile), or specific hydrogen bonding interactions with the solvent (acetic acid). Metadynamics simulations reveal a new chlorine addition mechanism explaining the autocatalytic effects of the reaction. The strength of combining static calculations and metadynamics simulations is highlighted, which provide complementary insight into chemical reactions in solvent.

Rational Design of Nanobody80 Loop Peptidomimetics: Towards Biased ß2 Adrenergic Receptor Ligands.

G protein-coupled receptors (GPCRs) play an important role in many cellular responses; as such, their mechanism of action is of utmost interest. To gain insight into the active conformation of GPCRs, the X-ray crystal structures of nanobody (Nb)-stabilized ß2 -adrenergic receptor (ß2 AR) have been reported. Nb80, in particular, is able to bind the intracellular G protein binding site of ß2 AR and stabilize the receptor in an active conformation. Within Nb80, the complementarity-determining region 3 (CDR3) is responsible for most of the binding interactions. Hence, we hypothesized that peptidomimetics of the CDR3 loop might be sufficient for binding to the receptor, inhibiting the interaction of ß2 AR with intracellular GPCR interacting proteins (e.g., G proteins). Based on previous crystallographic data, a set of peptidomimetics were synthesized that, similar to the Nb80 CDR3 loop, adopt a ß-hairpin conformation. Syntheses, conformational analysis, binding and functional in vitro assays, as well as internalization experiments, were performed. We demonstrate that peptidomimetics can structurally mimic the CDR3 loop of a nanobody and its function by inhibiting G protein coupling as measured by partial inhibition of cAMP production.

Influence of Solvation and Dynamics on the Mechanism and Kinetics of Nucleophilic Aromatic Substitution Reactions in Liquid Ammonia.

The role of the solvent and the influence of dynamics on the kinetics and mechanism of the SNAr reaction of several halonitrobenzenes in liquid ammonia, using both static calculations and dynamic ab initio molecular dynamics simulations, are investigated. A combination of metadynamics and committor analysis methods reveals how this reaction can change from a concerted, one-step mechanism in gas phase to a stepwise pathway, involving a metastable Meisenheimer complex, in liquid ammonia. This clearly establishes, among others, the important role of the solvent and highlights the fact that accurately treating solvation is of crucial importance to correctly unravel the reaction mechanism. It is indeed shown that H-bond formation of the reacting NH3 with the solvent drastically reduces the barrier of NH3 addition. The halide elimination step, however, is greatly facilitated by proton transfer from the reacting NH3 to the solvent. Furthermore, the free energy surface strongly depends on the halide substituent and the number of electron-withdrawing nitro substituents.

Cooperative dynamics of a DNA polymerase replicating complex.

Engineered DNA polymerases continue to be the workhorses of many applications in biotechnology, medicine and nanotechnology. However, the dynamic interplay between the enzyme and the DNA remains unclear. In this study, we performed an extensive replica exchange with flexible tempering (REFT) molecular dynamics simulation of the ternary replicating complex of the archaeal family B DNA polymerase from the thermophile Thermococcus gorgonarius, right before the chemical step. The convoluted dynamics of the enzyme are reducible to rigid-body motions of six subdomains. Upon binding to the enzyme, the DNA double helix conformation changes from a twisted state to a partially untwisted state. The twisted state displays strong bending motion, whereby the DNA oscillates between a straight and a bent conformation. The dynamics of double-stranded DNA are strongly correlated with rotations of the thumb toward the palm, which suggests an assisting role of the enzyme during DNA translocation. In the complex, the primer-template duplex displays increased preference for the B-DNA conformation at the n-2 and n-3 dinucleotide steps. Interactions at the primer 3' end indicate that Thr541 and Asp540 are the acceptors of the first proton transfer in the chemical step, whereas in the translocation step both residues hold the primer 3' terminus in the vicinity of the priming site, which is crucial for high processivity.

Normal mode analysis of Trp RNA binding attenuation protein: structure and collective motions.

The Trp RNA-binding protein (TRAP) has a toroidal topology and a perfect 11-fold symmetry, which makes it an excellent candidate for a vibrational study of elastic properties. Normal mode analysis in combination with correlation matrix calculations was used to detect collective low-frequency motions in TRAP. The results reveal the presence of highly correlated modes at the lower end of the spectrum, which directly reflect the annular and toroidal topology. The integral of the correlations over the low-frequency torsional part of the vibrational spectrum further demonstrates the relative rigidity of the 11 monomer building blocks of TRAP. The internal flexibility of each monomer and the effects of Trp-binding were also examined. The study clearly shows the determining influence of symmetry and topology on the elastic properties and also offers a detailed view on the Trp affinity of TRAP.

Tryptophan conformations associated with partial unfolding in ribonuclease T1.

The origin of the biexponential fluorescence decay of Trp in ribonuclease T1 under mildly destabilizing conditions, such as increased pH or temperature, or the presence of detergent, is still not understood. We have performed two extended replica-exchange molecular dynamics simulations to obtain a detailed representation of the native state at two protonation states corresponding to a high and low pH. At high pH, the appearance of partially unfolded states is evident. We found that this pH-induced destabilization originates from increased global repulsion as well as reduced local favorable electrostatic interactions and reduced H-bonding strength of His(27), His(40), and His(92). At high pH, alternative tryptophan rotamers appear and are linked to a distorted environment of the tryptophan, which also acts as a separate source of ground-state heterogeneity. The total population of these alternative conformations agrees well with the amplitude of the experimentally observed secondary fluorescence lifetime.

Water distribution at the electrified interface of deep eutectic solvents.

Deep eutectic solvents (DESs) are a new class of solvents with wider potential window than that of water and high electrochemical stability, making them potential candidates for a wide range of electrochemical systems. However, due to the hygroscopic nature of DESs, the presence of latent water is unavoidable. Therefore, understanding the interfacial structure and the electrosorption and distribution of residual water at the electrified interface is of great importance for the use of these solvents in electrochemical systems. Using atomistic molecular dynamics, we explore the electrosorption and distribution of different amounts of water in 1 : 2 choline chloride-urea DES (Reline) at the electrified graphene interface. We found that both the water distribution and the interfacial structure are sensitive to the electrification of the graphene electrode. As a result, it is found that for moderately charged electrodes, water shows a preferential asymmetric adsorption in the vicinity of the positively charged electrode, partly due to strong intermolecular interactions with anions through hydrogen bonds. In contrast, for highly charged electrodes, water adsorbs at both electrodes due to a strongly enhanced external electrostatic interaction between the electrodes and the water dipoles.

Correction: Water distribution at the electrified interface of deep eutectic solvents.

[This corrects the article DOI: 10.1039/C9NA00331B.].

How do rotameric conformations influence the time-resolved fluorescence of tryptophan in proteins? A perspective based on molecular modeling and quantum chemistry.

We discuss the dynamics of tryptophan rotamers in the context of the non-exponential fluorescence decay in proteins. The central question is: how does the ground-state conformational heterogeneity influence the time evolution of tryptophan fluorescence? This problem is examined here from the theoretical perspective. Three methods at different levels of theory, and with different scopes and computational requirements are reviewed. The Dead-end elimination method is limited to side-chain dynamics and provides an efficient way to detect the stable tryptophan rotamers in a protein. Its application to the study of heterogeneous emission characteristics is illustrated. Molecular dynamics is aimed at the full phase space of the macromolecule in solution, but must rely on classical force fields and laws of evolution. We examine to what extent the molecular mechanics paradigm yields sufficiently accurate thermodynamic results, and what are the possible kinetic implications. Finally Quantum Chemistry is the only theoretical method that allows a direct assessment of the excited states. It is necessarily restricted to small molecular systems, and thus must be used in a hybrid combination with classical methods and electrostatic models. So far understanding of the emitting state has greatly progressed as a result of these calculations, but the actual treatment of the photophysical decay processes at the quantum level has not yet really started.

Atomistic Insight into the Electrochemical Double Layer of Choline Chloride-Urea Deep Eutectic Solvents: Clustered Interfacial Structuring.

Green, stable, and wide electrochemical window deep eutectic solvents (DESs) are ideal candidates for electrochemical systems. However, despite several studies of their bulk properties, their structure and properties under electrified confinement have barely been investigated, which has hindered widespread use of these solvents in electrochemical applications. In this Letter, we explore the electrical double layer structure of 1:2 choline chloride-urea (Reline), with a particular focus on the electrosorption of the hydrogen bond donor on a graphene electrode using atomistic molecular dynamics simulations. We discovered that the interface is composed of a mixed layer of urea and counterions followed by a mixed charged clustered structure of all of the Reline components. This interfacial structuring is strongly dependent on the balance between intermolecular interactions and surface polarization. These results provide new insights into the electrical double layer structure of a new generation of electrolytes whose interfacial structure can be tuned at the molecular level.

Aromatic sulfonation with sulfur trioxide: mechanism and kinetic model.

Electrophilic aromatic sulfonation of benzene with sulfur trioxide is studied with ab initio molecular dynamics simulations in gas phase, and in explicit noncomplexing (CCl3F) and complexing (CH3NO2) solvent models. We investigate different possible reaction pathways, the number of SO3 molecules participating in the reaction, and the influence of the solvent. Our simulations confirm the existence of a low-energy concerted pathway with formation of a cyclic transition state with two SO3 molecules. Based on the simulation results, we propose a sequence of elementary reaction steps and a kinetic model compatible with experimental data. Furthermore, a new alternative reaction pathway is proposed in complexing solvent, involving two SO3 and one CH3NO2.

The harmonic analysis of cylindrically symmetric proteins: a comparison of Dronpa and a DNA sliding clamp.

The harmonic analysis of two types of proteins with cylindrical symmetry is performed by the Standard Force Field Normal Mode Analysis and by the elastic network model. For both proteins the global elastic modes are assigned to their characteristic topologies. Dronpa is a rigid ß-barrel structure, presenting the twisting, bending and breathing motion of a cylindrical rod. The ß sliding clamp of Escherichia coli is a hexagonal ß-wheel, consisting of rigid segments. In its spectrum four classes of vibrations are identified which are characteristic of an elastic torus. Correlation diagrams and RMSF analysis are compared. The results provide not only a comprehensive validation of the use of both methods to describe the elastic behavior according to the low-frequency normal modes, but also depict the correlated motions of ß-barrel and ß-wheel proteins. The harmonic flexibility of the Dronpa protein is compared to the principal components of molecular dynamics (MD) simulation. A functionally important localized cleft opening mode is found, which is not detected by harmonic analysis.

Improved Replica Exchange Method for Native-State Protein Sampling.

We present a new replica exchange method, designed for optimal native state protein sampling in explicit solvent, called replica exchange with flexible tempering (REFT). The method was built upon the recently introduced replica exchange with solute tempering (REST). The potential function is adapted to direct the conformational search toward interdomain movements and the flexible portions of the protein. We demonstrate the improved sampling efficiency of REFT compared to the original REST for the bacteriophage T4 lysozyme.

Structural basis for the role of LYS220 as proton donor for nucleotidyl transfer in HIV-1 reverse transcriptase.

Biochemical studies by Castro et al. have recently revealed a crucial role for a general acid in the catalysis of nucleic acid transfer in distinct classes of polymerases. For HIV-RT LYS220 was identified as proton donor. This was unanticipated from a structural point of view, since in all ternary crystal structures of HIV-RT LYS220 are too distant from the active site to fulfill this role. In this work molecular dynamics simulations were used to reveal the dynamics of HIV-RT and to provide structural evidence for the role of LYS220. During a 1µs molecular dynamics simulation LYS220 migrates toward the active site and occupies several positions enabling direct and water mediated proton transfer towards pyrophosphate. A combination of quantum mechanical and molecular mechanics methods was used to validate the different modes of interaction.

Structure-based site of metabolism prediction for cytochrome P450 2D6.

Realistic representation of protein flexibility in biomolecular simulations remains an unsolved fundamental problem and is an active area of research. The high flexibility of the cytochrome P450 2D6 (CYP2D6) active site represents a challenge for accurate prediction of the preferred binding mode and site of metabolism (SOM) for compounds metabolized by this important enzyme. To account for this flexibility, we generated a large ensemble of unbiased CYP2D6 conformations, to which small molecule substrates were docked to predict their experimentally observed SOM. SOM predictivity was investigated as a function of the number of protein structures, the scoring function, the SOM-heme cutoff distance used to distinguish metabolic sites, and intrinsic reactivity. Good SOM predictions for CYP2D6 require information from the protein. A critical parameter is the distance between the heme iron and the candidate site of metabolism. The best predictions were achieved with cutoff distances consistent with the chemistry relevant to CYP2D6 metabolism. Combination of the new ensemble-based docking method with estimated intrinsic reactivities of substrate sites considerably improved the predictivity of the model. Testing on an independent set of substrates yielded area under curve values as high as 0.93, validating our new approach.

How Is cis-trans Isomerization Controlled in Dronpa Mutants? A Replica Exchange Molecular Dynamics Study.

The reversibly photoactivatable green fluorescent protein analog Dronpa holds great promise as a marker for various new cellular imaging applications. Using a replica exchange method which combines both Hamiltonian and temperature exchanges, the ground-state dynamics of Dronpa and two mutants with increased switching kinetics, Val157Gly and Met159Thr, were compared. The dominant chromophore state was found to be the cis isomer in all three proteins. The simulation data suggest that both mutations strongly increase the chromophore flexibility and cis-trans isomerization rate. We identify three key amino acids, Val157, Met159, and Phe173, which are able to impede the bottom hula-twist transition path, depending on their position and rotameric state. We believe our insights will help to understand the switching process and provide useful information for the design of new variants with improved fluorescence properties.

Tryptophan rotamers as evidenced by X-ray, fluorescence lifetimes, and molecular dynamics modeling.

We investigated the native-state dynamics of the Bacillus caldolyticus cold-shock protein mutant Bc-Csp L66E, using fluorescence and appropriate molecular dynamics methods. Two fluorescence lifetimes were found, the amplitudes of which agree very well with tryptophan rotamer populations, obtained from parallel tempering calculations. Rotamer lifetimes were predicted by transition-state theory from high-temperature simulations. Transition pathways were extracted from the transition rates between individual rotameric states. The molecular dynamics also reveal the loop fluctuations in the native state.