Evidence of H/D Exchange within Metal-Adducted Carbohydrates after Ion/Ion-Dissociation Reactions.

Tandem mass spectrometry (MS/MS) using fragmentation has become one of the most effective methods for gaining sequence and structural information on biomolecules. Ion/ion reactions are competitive reactions, where either proton transfer (PT) or electron transfer (ET) can occur from interactions between multiply charged cations and singly charged anions. Utilizing ion/ion reactions with fluoranthene has offered a unique method of fragment formation for the structural elucidation of biomolecules. Fluoranthene is considered an ideal anion reagent because it selectively causes electron-transfer dissociation (ETD) and minimizes PT when interacting with peptides. However, limited investigations have sought to understand how fluoranthene─the primary, commercially available anion reagent─interacts with other biomolecules. Here, we apply deuterium labeling to investigate ion/ion reaction mechanisms between fluoranthene and divalent, metal-adducted carbohydrates (Ca2+, Mg2+, Co2+, and Ni2+). Deuterium labeling of carbohydrates allowed us to observe evidence of hydrogen/deuterium exchange (HDX) occurring after ion/ion dissociation reactions. The extent of deuterium loss is dependent on several factors, including the physical properties of the metal ion and the fragment structure. Based on the deuterium labeling data, we have proposed ETD, PTD, and intermolecular PT─also described as HDX─mechanisms. This research provides a fundamental perspective of ion/ion and ion/molecule reaction mechanisms and illustrates properties that impact ion/ion and ion/molecule reactions for carbohydrates. Together, this could improve the capability to distinguish complex and heterogeneous biomolecules, such as carbohydrates.

Impact of protein conformational diversity on AlphaFold predictions.

MOTIVATION: After the outstanding breakthrough of AlphaFold in predicting protein 3D models, new questions appeared and remain unanswered. The ensemble nature of proteins, for example, challenges the structural prediction methods because the models should represent a set of conformers instead of single structures. The evolutionary and structural features captured by effective deep learning techniques may unveil the information to generate several diverse conformations from a single sequence. Here, we address the performance of AlphaFold2 predictions obtained through ColabFold under this ensemble paradigm. RESULTS: Using a curated collection of apo-holo pairs of conformers, we found that AlphaFold2 predicts the holo form of a protein in ∼70% of the cases, being unable to reproduce the observed conformational diversity with the same error for both conformers. More importantly, we found that AlphaFold2's performance worsens with the increasing conformational diversity of the studied protein. This impairment is related to the heterogeneity in the degree of conformational diversity found between different members of the homologous family of the protein under study. Finally, we found that main-chain flexibility associated with apo-holo pairs of conformers negatively correlates with the predicted local model quality score plDDT, indicating that plDDT values in a single 3D model could be used to infer local conformational changes linked to ligand binding transitions. AVAILABILITY AND IMPLEMENTATION: Data and code used in this manuscript are publicly available at https://gitlab.com/sbgunq/publications/af2confdiv-oct2021. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Impact of the core on the inter-branch exciton exchange in dendrimers.

Organic dendrimers with π conjugated systems are capable of capturing solar energy as a renewable source for human use. Nonetheless, further study regarding the relationship between the structure and the energy transfer mechanism in these types of molecules is still necessary. In this work, nonadiabatic excited state molecular dynamics (NEXMD) were carried out to study the intra- and inter-branch exciton migration in two tetra-branched dendrimers, C(dSSB)4 and Ad(BuSSB)4, which differ in their respective carbon and adamantane core. Both systems undergo a ladder decay mechanism between excited states, with back-and-forth transitions between S1 and S2. Despite presenting very similar absorption-emission spectra, differences in the photoinduced energy relaxation are observed. The size of the core impacts the inter-branch energy exchange and transient exciton localization/delocalization, which ultimately condition the relative energy relaxation rates, being faster in Ad(BuSSB)4 with respect to C(dSSB)4. Nevertheless, the photoinduced processes lead to a progressive final exciton-self-trapping in one of the branches of both dendrimers, which is a desirable feature in organic photovoltaic applications. Our results can inspire the design of more efficient dendrimers with the desired magnitude of inter-branch exciton exchange and localization/delocalization according to changes in their core.

RepeatsDB in 2021: improved data and extended classification for protein tandem repeat structures.

The RepeatsDB database (URL: https://repeatsdb.org/) provides annotations and classification for protein tandem repeat structures from the Protein Data Bank (PDB). Protein tandem repeats are ubiquitous in all branches of the tree of life. The accumulation of solved repeat structures provides new possibilities for classification and detection, but also increasing the need for annotation. Here we present RepeatsDB 3.0, which addresses these challenges and presents an extended classification scheme. The major conceptual change compared to the previous version is the hierarchical classification combining top levels based solely on structural similarity (Class > Topology > Fold) with two new levels (Clan > Family) requiring sequence similarity and describing repeat motifs in collaboration with Pfam. Data growth has been addressed with improved mechanisms for browsing the classification hierarchy. A new UniProt-centric view unifies the increasingly frequent annotation of structures from identical or similar sequences. This update of RepeatsDB aligns with our commitment to develop a resource that extracts, organizes and distributes specialized information on tandem repeat protein structures.

Analysis of changes of cavity volumes in predefined directions of protein motions and cavity flexibility.

Dynamics of protein cavities associated with protein fluctuations and conformational plasticity is essential for their biological function. NMR ensembles, molecular dynamics (MD) simulations, and normal mode analysis (NMA) provide appropriate frameworks to explore functionally relevant protein dynamics and cavity changes relationships. Within this context, we have recently developed analysis of null areas (ANA), an efficient method to calculate cavity volumes. ANA is based on a combination of algorithms that guarantees its robustness against numerical differentiations. This is a unique feature with respect to other methods. Herein, we present an updated and improved version that expands it use to quantify changes in cavity features, like volume and flexibility, due to protein structural distortions performed on predefined biologically relevant directions, for example, directions of largest contribution to protein fluctuations (principal component analysis [PCA modes]) obtained by MD simulations or ensembles of NMR structures, collective NMA modes or any other direction of motion associated with specific conformational changes. A web page has been developed where its facilities are explained in detail. First, we show that ANA can be useful to explore gradual changes of cavity volume and flexibility associated with protein ligand binding. Secondly, we perform a comparison study of the extent of variability between protein backbone structural distortions, and changes in cavity volumes and flexibilities evaluated for an ensemble of NMR active and inactive conformers of the epidermal growth factor receptor structures. Finally, we compare changes in size and flexibility between sets of NMR structures for different homologous chains of dynein.

Non-adiabatic Excited-State Molecular Dynamics: Theory and Applications for Modeling Photophysics in Extended Molecular Materials.

Optically active molecular materials, such as organic conjugated polymers and biological systems, are characterized by strong coupling between electronic and vibrational degrees of freedom. Typically, simulations must go beyond the Born-Oppenheimer approximation to account for non-adiabatic coupling between excited states. Indeed, non-adiabatic dynamics is commonly associated with exciton dynamics and photophysics involving charge and energy transfer, as well as exciton dissociation and charge recombination. Understanding the photoinduced dynamics in such materials is vital to providing an accurate description of exciton formation, evolution, and decay. This interdisciplinary field has matured significantly over the past decades. Formulation of new theoretical frameworks, development of more efficient and accurate computational algorithms, and evolution of high-performance computer hardware has extended these simulations to very large molecular systems with hundreds of atoms, including numerous studies of organic semiconductors and biomolecules. In this Review, we will describe recent theoretical advances including treatment of electronic decoherence in surface-hopping methods, the role of solvent effects, trivial unavoided crossings, analysis of data based on transition densities, and efficient computational implementations of these numerical methods. We also emphasize newly developed semiclassical approaches, based on the Gaussian approximation, which retain phase and width information to account for significant decoherence and interference effects while maintaining the high efficiency of surface-hopping approaches. The above developments have been employed to successfully describe photophysics in a variety of molecular materials.

Exciton-vibrational dynamics induces efficient self-trapping in a substituted nanoring.

Cycloparaphenylenes, being the smallest segments of carbon nanotubes, have emerged as prototypes of the simplest carbon nanohoops. Their unique structure-dynamics-optical properties relationships have motivated a wide variety of synthesis of new related nanohoop species. Studies of how chemical changes, introduced in these new materials, lead to systems with new structural, dynamics and optical properties, expand their functionalities for optoelectronics applications. Herein, we study the effect that conjugation extension of a cycloparaphenylene through the introduction of a satellite tetraphenyl substitution has on its structural and dynamical properties. Our non-adiabatic excited state molecular dynamics simulations suggest that this substitution accelerates the electronic relaxation from the high-energy band to the lowest excited state. This is partially due to efficient conjugation achieved between specific phenyl units as introduced by the tetraphenyl substitution. We observe a particular exciton redistribution during relaxation, in which the tetraphenyl substitution plays a significant role. As a result, an efficient inter-band energy transfer takes place. Besides, the observed phonon-exciton interplay induces a significant exciton self-trapping. Our results encourage and guide the future studies of new phenyl substitutions in carbon nanorings with desired optoelectronic properties.

Vibronic Photoexcitation Dynamics of Perylene Diimide: Computational Insights.

Perylene diimide (PDI) represents a prototype material for organic optoelectronic devices because of its strong optical absorbance, chemical stability, efficient energy transfer, and optical and chemical tunability. Herein, we analyze in detail the vibronic relaxation of its photoexcitation using nonadiabatic excited-state molecular dynamics simulations. We find that after the absorption of a photon, which excites the electron to the second excited state, S2, induced vibronic dynamics features persistent modulations in the spatial localization of electronic and vibrational excitations. These energy exchanges are dictated by strong vibronic couplings that overcome structural disorders and thermal fluctuations. Specifically, the electronic wavefunction periodically swaps between localizations on the right and left sides of the molecule. Within 1 ps of such dynamics, a nonradiative transition to the lowest electronic state, S1, takes place, resulting in a complete delocalization of the wavefunction. The observed vibronic dynamics emerges following the electronic energy deposition in the direction that excites a combination of two dominant vibrational normal modes. This behavior is maintained even with a chemical substitution that breaks the symmetry of the molecule. We believe that our findings elucidate the nature of the complex dynamics of the optically excited states and, therefore, contribute to the development of tunable functionalities of PDIs and their derivatives.

Environmental surveillance of Legionella in tourist facilities of the Balearic Islands, Spain, 2006 to 2010 and 2015 to 2018.

BackgroundLegionnaires' disease is a respiratory illness often associated with hotels and travel. Spain is a major tourist destination and one of the European countries with most cases of Legionnaires' disease , both community- and travel-associated. However, the prevalence of Legionella in tourist facilities is unknown.AimThe present investigation aimed to survey the tourist facilities in the Balearic Islands, Spain, for Legionella prevalence.MethodsWe visited tourist facilities in the Balearic Islands in two different periods (2006-2010 and 2015-2018) and took water samples following national and international guidelines. Legionella was investigated by culture methods following international standards (ISO 11731:1998).ResultsWe evaluated 13,472 samples from 465 facilities. Bacteria of the Legionella genus were detected in 65.4% of the surveyed facilities. Contamination of the facilities was significantly higher during the second decade (54.5 vs 78.6%). The most frequent colonisers were L. pneumophila serogroup 2-14. We detected the pathogen in 15.9% and 6.9% of hot and cold water distribution systems samples, respectively. The Legionella contamination rate in cold water systems samples was higher when free chlorine levels were < 0.2 mg/L and at > 25 °C temperatures, while in the hot water systems samples, the contamination rate was higher at < 50 °C. Of the samples from hot tubs, 10.9% were contaminated.ConclusionLegionella prevalence in hotels in the Balearic Islands was high but the contamination rates depended on the installations. Corrective measures are still needed to improve Legionella control.

Excitation Energy Transfer between bodipy Dyes in a Symmetric Molecular Excitonic Seesaw.

We examine the redistribution of energy between electronic and vibrational degrees of freedom that takes place between a π-conjugated oligomer, a phenylene-butadiynylene, and two identical boron-dipyrromethene (bodipy) end-caps using femtosecond transient absorption spectroscopy, single-molecule spectroscopy, and nonadiabatic excited-state molecular dynamics (NEXMD) modeling techniques. The molecular structure represents an excitonic seesaw in that the excitation energy on the oligomer backbone can migrate to either one end-cap or the other, but not to both. The NEXMD simulations closely reproduce the characteristic time scale for redistribution of electronic and vibrational energy of 2.2 ps and uncover the vibrational modes contributing to the intramolecular relaxation. The calculations indicate that the dihedral angle between the bodipy dye and the oligomer change upon excitation of the oligomer. Single-molecule experiments reveal a difference in photoluminescence lifetime of the bodipy dyes depending on whether they are excited by direct absorption or by redistribution of energy from the backbone. This difference in lifetime may be attributed to the difference in dihedral angle. The simulations also suggest that a strong coupling can occur between the two end-caps, giving rise to a reversible shuttling of excitation energy between them. Strong coupling should lead to a pronounced loss in polarization memory of the fluorescence since the oligomer backbone tends to be slightly distorted and the two bodipy transition dipoles have different orientations. A sensitive single-molecule technique is presented to test for such coupling. However, although redistribution of electronic and vibrational energy between the end-caps can occur, it appears to be unidirectional and irreversible, suggesting that an additional localization mechanism is at play which is, as yet, not fully accounted for in the simulations.

Integrated Nanophotonic Waveguide-Based Devices for IR and Raman Gas Spectroscopy.

On-chip devices for absorption spectroscopy and Raman spectroscopy have been developing rapidly in the last few years, triggered by the growing availability of compact and affordable tunable lasers, detectors, and on-chip spectrometers. Material processing that is compatible with mass production has been proven to be capable of long low-loss waveguides of sophisticated designs, which are indispensable for high-light-analyte interactions. Sensitivity and selectivity have been further improved by the development of sorbent cladding. In this review, we discuss the latest advances and challenges in the field of waveguide-enhanced Raman spectroscopy (WERS) and waveguide infrared absorption spectroscopy (WIRAS). The development of integrated light sources and detectors toward miniaturization will be presented, together with the recent advances on waveguides and cladding to improve sensitivity. The latest reports on gas-sensing applications and main configurations for WERS and WIRAS will be described, and the most relevant figures of merit and limitations of different sensor realizations summarized.

Exploring Conformational Space with Thermal Fluctuations Obtained by Normal-Mode Analysis.

Proteins in their native states can be represented as ensembles of conformers in dynamical equilibrium. Thermal fluctuations are responsible for transitions between these conformers. Normal-modes analysis (NMA) using elastic network models (ENMs) provides an efficient procedure to explore global dynamics of proteins commonly associated with conformational transitions. In the present work, we present an iterative approach to explore protein conformational spaces by introducing structural distortions according to their equilibrium dynamics at room temperature. The approach can be used either to perform unbiased explorations of conformational space or to explore guided pathways connecting two different conformations, e.g., apo and holo forms. In order to test its performance, four proteins with different magnitudes of structural distortions upon ligand binding have been tested. In all cases, the conformational selection model has been confirmed and the conformational space between apo and holo forms has been encompassed. Different strategies have been tested that impact on the efficiency either to achieve a desired conformational change or to achieve a balanced exploration of the protein conformational multiplicity.

Photoexcitation dynamics in perylene diimide dimers.

We utilize first-principles theory to investigate photo-induced excited-state dynamics of functionalized perylene diimide. This class of materials is highly suitable for solar energy conversion because of the strong optical absorbance, efficient energy transfer, and chemical tunability. We couple time-dependent density functional theory to a recently developed time-resolved non-adiabatic dynamics approach based on a semi-empirical description. By studying the monomer and dimer, we focus on the role stacking plays on the time-scales associated with excited-state non-radiative relaxation from a high excitonic state to the lowest energy exciton. We predict that the time-scale for energy conversion in the dimer is significantly faster than that in the monomer when equivalent excited states are accounted for. Additionally, for the dimer, the decay from the second to the nearly degenerate lowest energy excited-state involves two time-scales: a rapid decay on the order of ∼10 fs followed by a slower decay of ∼100 fs. Analysis of the spatial localization of the electronic transition density during the internal conversion process points out the existence of localized states on individual monomers, indicating that the strength of thermal fluctuations exceeds electronic couplings between the states such that the exciton hops between localized states throughout the simulation.

Experimental and theoretical study of energy transfer in a chromophore triad: What makes modeling dynamics successful?

Simulation of electronic dynamics in realistically large molecular systems is a demanding task that has not yet achieved the same level of quantitative prediction already realized for its static counterpart. This is particularly true for processes occurring beyond the Born-Oppenheimer regime. Non-adiabatic molecular dynamics (NAMD) simulations suffer from two convoluted sources of error: numerical algorithms for dynamics and electronic structure calculations. While the former has gained increasing attention, particularly addressing the validity of ad hoc methodologies, the effect of the latter remains relatively unexplored. Indeed, the required accuracy for electronic structure calculations to reach quantitative agreement with experiment in dynamics may be even more strict than that required for static simulations. Here, we address this issue by modeling the electronic energy transfer in a donor-acceptor-donor (D-A-D) molecular light harvesting system using fewest switches surface hopping NAMD simulations. In the studied system, time-resolved experimental measurements deliver complete information on spectra and energy transfer rates. Subsequent modeling shows that the calculated electronic transition energies are "sufficiently good" to reproduce experimental spectra but produce over an order of magnitude error in simulated dynamical rates. We further perform simulations using artificially shifted energy gaps to investigate the complex relationship between transition energies and modeled dynamics to understand factors affecting non-radiative relaxation and energy transfer rates.

Chemical Stability of Mesoporous Oxide Thin Film Electrodes under Electrochemical Cycling: from Dissolution to Stabilization.

Mesoporous oxide thin films (MOTF) present very high surface areas and highly controlled monodisperse pores in the nanometer range. These features spurred their possible applications in separation membranes and permselective electrodes. However, their performance in real applications is limited by their reactivity. Here, we perform a basic study of the stability of MOTF toward dissolution in aqueous media using a variety of characterization techniques. In particular, we focus in their stability behavior under the influence of ionic strength, adsorption of electrochemical probes, and applied electrode potential. Mesoporous silica thin films present a limited chemical stability after electrochemical cycling, particularly under high ionic strength, due to their high specific surface area and the interactions between the electrochemical probes and the surface. In contrast, TiO2 or Si0.9Zr0.1O2 matrices present higher stability; thus, they are an adequate alternative to produce accessible, sensitive, and robust permselective electrodes or membranes that perform under a wide variety of conditions.

Network analysis of dynamically important residues in protein structures mediating ligand-binding conformational changes.

According to the generalized conformational selection model, ligand binding involves the co-existence of at least two conformers with different ligand-affinities in a dynamical equilibrium. Conformational transitions between them should be guaranteed by intramolecular vibrational dynamics associated to each conformation. These motions are, therefore, related to the biological function of a protein. Positions whose mutations are found to alter these vibrations the most can be defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. In a previous study, we have shown that these positions are evolutionarily conserved. They correspond to buried aliphatic residues mostly localized in regular structured regions of the protein like ß-sheets and α-helices. In the present paper, we perform a network analysis of these key positions for a large dataset of paired protein structures in the ligand-free and ligand-bound form. We observe that networks of interactions between these key positions present larger and more integrated networks with faster transmission of the information. Besides, networks of residues result that are robust to conformational changes. Our results reveal that the conformational diversity of proteins seems to be guaranteed by a network of strongly interconnected key positions rather than individual residues.

Dynamics fingerprints of active conformers of epidermal growth factor receptor kinase.

Epidermal growth factor receptor (EGFR) is a prototypical cell-surface receptor that plays a key role in the regulation of cellular signaling, proliferation and differentiation. Mutations of its kinase domain have been associated with the development of a variety of cancers and, therefore, it has been the target of drug design. Single amino acid substitutions (SASs) in this domain have been proven to alter the equilibrium of pre-existing conformer populations. Despite the advances in structural descriptions of its so-called active and inactive conformations, the associated dynamics aspects that characterize them have not been thoroughly studied yet. As the dynamic behaviors and molecular motions of proteins are important for a complete understanding of their structure-function relationships we present a novel procedure, using (or based on) normal mode analysis, to identify the collective dynamics shared among different conformers in EGFR kinase. The method allows the comparison of patterns of low-frequency vibrational modes defining representative directions of motions. Our procedure is able to emphasize the main similarities and differences between the collective dynamics of different conformers. In the case of EGFR kinase, two representative directions of motions have been found as dynamics fingerprints of the active conformers. Protein motion along both directions reveals to have a significant impact on the cavity volume of the main pocket of the active site. Otherwise, the inactive conformers exhibit a more heterogeneous distribution of collective motions. © 2018 Wiley Periodicals, Inc.

Conformational diversity analysis reveals three functional mechanisms in proteins.

Protein motions are a key feature to understand biological function. Recently, a large-scale analysis of protein conformational diversity showed a positively skewed distribution with a peak at 0.5 Å C-alpha root-mean-square-deviation (RMSD). To understand this distribution in terms of structure-function relationships, we studied a well curated and large dataset of ~5,000 proteins with experimentally determined conformational diversity. We searched for global behaviour patterns studying how structure-based features change among the available conformer population for each protein. This procedure allowed us to describe the RMSD distribution in terms of three main protein classes sharing given properties. The largest of these protein subsets (~60%), which we call "rigid" (average RMSD = 0.83 Å), has no disordered regions, shows low conformational diversity, the largest tunnels and smaller and buried cavities. The two additional subsets contain disordered regions, but with differential sequence composition and behaviour. Partially disordered proteins have on average 67% of their conformers with disordered regions, average RMSD = 1.1 Å, the highest number of hinges and the longest disordered regions. In contrast, malleable proteins have on average only 25% of disordered conformers and average RMSD = 1.3 Å, flexible cavities affected in size by the presence of disordered regions and show the highest diversity of cognate ligands. Proteins in each set are mostly non-homologous to each other, share no given fold class, nor functional similarity but do share features derived from their conformer population. These shared features could represent conformational mechanisms related with biological functions.

An ab initio multiple cloning approach for the simulation of photoinduced dynamics in conjugated molecules.

We present a new implementation of the Ab Initio Multiple Cloning (AIMC) method, which is applied for non-adiabatic excited-state molecular dynamics simulations of photoinduced processes in conjugated molecules. Within our framework, the multidimensional wave-function is decomposed into a superposition of a number of Gaussian coherent states guided by Ehrenfest trajectories that are suited to clone and swap their electronic amplitudes throughout the simulation. New generalized cloning criteria are defined and tested. Because of sharp changes of the electronic states, which are common for conjugated polymers, the electronic parts of the Gaussian coherent states are represented in the Time Dependent Diabatic Basis (TDDB). The input to these simulations in terms of the excited-state energies, gradients and non-adiabatic couplings, is calculated on-the-fly using the Collective Electron Oscillator (CEO) approach. As a test case, we consider the photoinduced unidirectional electronic and vibrational energy transfer between two- and three-ring linear poly(phenylene ethynylene) units linked by meta-substitution. The effects of the cloning procedure on electronic and vibrational coherence, relaxation and unidirectional energy transfer between dendritic branches are discussed.

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.