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Understanding the dynamics of biomolecular complexes, e.g., of protein-ligand (un)binding, requires the comprehension of paths such systems take between metastable states. In MD simulations, paths are usually not observable per se, but they need to be inferred from simulation trajectories. Here, we present a novel approach to cluster trajectories based on a community detection algorithm that necessitates only the definition of a single parameter. The unbinding of the streptavidin-biotin complex is used as a benchmark system and the A 2a adenosine receptor in complex with the inhibitor ZM241385 as an elaborate application. We demonstrate how such clusters of trajectories correspond to pathways and how the approach helps in the identification of reaction coordinates for a considered (un)binding process.

The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of proliferation, the ingression of embryonic epiblast cells at the primitive streak, and their priming toward primitive streak fates. How these different events are coordinated remains unknown. Here, we developed and characterized a 3D culture of self-renewing mouse embryonic cells that captures the main transcriptional and architectural features of the early gastrulating mouse epiblast. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation-induced crowding triggers delamination of cells that express high levels of the apical polarity protein aPKC. Upon delamination, cells become more sensitive to Wnt signaling and upregulate the expression of primitive streak markers such as Brachyury. This mechanistic coupling between ingression and differentiation ensures that the right cell types become specified at the right place during embryonic development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 MRC Laboratory of Molecular Biology. Published by Elsevier Inc. All rights reserved.)

A novel mixed quantum-classical approach to simulating nonadiabatic dynamics of molecules at metal surfaces is presented. The method combines the numerically exact hierarchical equations of motion approach for the quantum electronic degrees of freedom with Langevin dynamics for the classical degrees of freedom, namely, low-frequency vibrational modes within the molecule. The approach extends previous mixed quantum-classical methods based on Langevin equations to models containing strong electron-electron or quantum electronic-vibrational interactions, while maintaining a nonperturbative and non-Markovian treatment of the molecule-metal coupling. To demonstrate the approach, nonequilibrium transport observables are calculated for a molecular nanojunction containing strong interactions., (© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).)

To sample rare events, dissipation-corrected targeted molecular dynamics (dcTMD) applies a constant velocity constraint along a one-dimensional reaction coordinate s , which drives an atomistic system from an initial state into a target state. Employing a cumulant approximation of Jarzynski's identity, the free energy Δ G ( s ) is calculated from the mean external work and dissipated work of the process. By calculating the friction coefficient Γ( s ) from the dissipated work, in a second step, the equilibrium dynamics of the process can be studied by propagating a Langevin equation. While so far dcTMD has been mostly applied to study the unbinding of protein-ligand complexes, here its applicability to rare conformational transitions within a protein and the prediction of their kinetics are investigated. As this typically requires the introduction of multiple collective variables { x j } = x , a theoretical framework is outlined to calculate the associated free energy Δ G ( x ) and friction Γ ( x ) from dcTMD simulations along coordinate s . Adopting the α-β transition of alanine dipeptide as well as the open-closed transition of T4 lysozyme as representative examples, the virtues and shortcomings of dcTMD to predict protein conformational transitions and the related kinetics are studied.

Protein dynamics have been investigated on a wide range of time scales. Nano- and picosecond dynamics have been assigned to local fluctuations, while slower dynamics have been attributed to larger conformational changes. However, it is largely unknown how fast (local) fluctuations can lead to slow global (allosteric) changes. Here, fast molecule-spanning dynamics on the 100 to 200 ns time scale in the heat shock protein 90 (Hsp90) are shown. Global real-space movements are assigned to dynamic modes on this time scale, which is possible by a combination of single-molecule fluorescence, quasi-elastic neutron scattering and all-atom molecular dynamics (MD) simulations. The time scale of these dynamic modes depends on the conformational state of the Hsp90 dimer. In addition, the dynamic modes are affected to various degrees by Sba1, a co-chaperone of Hsp90, depending on the location within Hsp90, which is in very good agreement with MD simulations. Altogether, this data is best described by fast molecule-spanning dynamics, which precede larger conformational changes in Hsp90 and might be the molecular basis for allostery. This integrative approach provides comprehensive insights into molecule-spanning dynamics on the nanosecond time scale for a multi-domain protein., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

The effect of an externally applied directional force on molecular friction is so far poorly understood. Here, we study the force-driven dissociation of the ligand-protein complex biotin-streptavidin and identify anisotropic friction as a not yet described type of molecular friction. Using AFM-based stereographic single molecule force spectroscopy and targeted molecular dynamics simulations, we find that the rupture force and friction for biotin-streptavidin vary with the pulling angle. This observation holds true for friction extracted from Kramers' rate expression and by dissipation-corrected targeted molecular dynamics simulations based on Jarzynski's identity. We rule out ligand solvation and protein-internal friction as sources of the angle-dependent friction. Instead, we observe a heterogeneity in free energy barriers along an experimentally uncontrolled orientation parameter, which increases the rupture force variance and therefore the overall friction. We anticipate that anisotropic friction needs to be accounted for in a complete understanding of friction in biomolecular dynamics and anisotropic mechanical environments.

The prediction of drug-target binding and unbinding kinetics that occur on time scales between milliseconds and several hours is a prime challenge for biased molecular dynamics simulation approaches. This Perspective gives a concise summary of the theory and the current state-of-the-art of such predictions via biased simulations, of insights into the molecular mechanisms defining binding and unbinding kinetics as well as of the extraordinary challenges predictions of ligand kinetics pose in comparison to binding free energy predictions.

Protein-ligand (un)binding simulations are a recent focus of biased molecular dynamics simulations. Such binding and unbinding can occur via different pathways in and out of a binding site. Here, we present a theoretical framework on how to compute kinetics along separate paths and on how to combine the path-specific rates into global binding and unbinding rates for comparison with experimental results. Using dissipation-corrected targeted molecular dynamics in combination with temperature-boosted Langevin equation simulations [S. Wolf et al., Nat. Commun. 11, 2918 (2020)] applied to a two-dimensional model and the trypsin-benzamidine complex as test systems, we assess the robustness of the procedure and discuss the aspects of its practical applicability to predict multisecond kinetics of complex biomolecular systems.

Photoproteins such as bacteriorhodopsin (bR) and rhodopsin (Rho) need to effectively dissipate photoinduced excess energy to prevent themselves from damage. Another well-studied seven transmembrane (TM) helices protein is the β 2 adrenergic receptor (β 2 AR), a G protein-coupled receptor for which energy dissipation paths have been linked with allosteric communication. To study the vibrational energy transport in the active and inactive states of these proteins, a master equation approach [ J. Chem. Phys. 2020 , 152 , 045103] is employed, which uses scaling rules that allow us to calculate energy transport rates solely based on the protein structure. Despite their overall structural similarity, the three 7TM proteins reveal quite different strategies to redistribute excess energy. While bR quickly removes the energy using the TM7 helix as a "lightning rod", Rho exhibits a rather poor energy dissipation, which might eventually require the hydrolysis of the Schiff base between the protein and the retinal chromophore to prevent overheating. Heating the ligand adrenaline of β 2 AR, the resulting energy transport network of the protein is found to change significantly upon switching from the active state to the inactive state. While the energy flow may highlight aspects of the inter-residue couplings of β 2 AR, it seems not particularly suited to explain allosteric phenomena.

While allostery is of paramount importance for protein signaling and regulation, the underlying dynamical process of allosteric communication is not well understood. The PDZ3 domain represents a prime example of an allosteric single-domain protein, as it features a well-established long-range coupling between the C-terminal α 3 -helix and ligand binding. In an intriguing experiment, Hamm and co-workers employed photoswitching of the α 3 -helix to initiate a conformational change of PDZ3 that propagates from the C-terminus to the bound ligand within 200 ns. Performing extensive nonequilibrium molecular dynamics simulations, the modeling of the experiment reproduces the measured time scales and reveals a detailed picture of the allosteric communication in PDZ3. In particular, a correlation analysis identifies a network of contacts connecting the α 3 -helix and the core of the protein, which move in a concerted manner. Representing a one-step process and involving direct α 3 -ligand contacts, this cooperative transition is considered as the elementary step in the propagation of conformational change.

We present two methods to reveal protein-ligand unbinding mechanisms in biased unbinding simulations by clustering trajectories into ensembles representing unbinding paths. The first approach is based on a contact principal component analysis for reducing the dimensionality of the input data, followed by identification of unbinding paths and training a machine learning model for trajectory clustering. The second approach clusters trajectories according to their pairwise mean Euclidean distance employing the neighbor-net algorithm, which takes into account input data bias in the distances set and is superior to dendrogram construction. Finally, we describe a more complex case where the reaction coordinate relevant for path identification is a single intraligand hydrogen bond, highlighting the challenges involved in unbinding path reaction coordinate detection.

Allosteric communication between distant protein sites represents a key mechanism of biomolecular regulation and signal transduction. Compared to other processes such as protein folding, however, the dynamical evolution of allosteric transitions is still not well understood. As an example of allosteric coupling between distant protein regions, we consider the global open-closed motion of the two domains of T4 lysozyme, which is triggered by local motion in the hinge region. Combining extensive molecular dynamics simulations with a correlation analysis of interresidue contacts, we identify a network of interresidue distances that move in a concerted manner. The cooperative process originates from a cogwheel-like motion of the hydrophobic core in the hinge region, which constitutes an evolutionary conserved and flexible transmission network. Through rigid contacts and the protein backbone, the small local changes of the hydrophobic core are passed on to the distant terminal domains and lead to the emergence of a rare global conformational transition. As in an Ising-type model, the cooperativity of the allosteric transition can be explained via the interaction of local fluctuations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The friction coefficient of fluids may become a function of the velocity at increased external driving. This non-Newtonian behavior is of general theoretical interest and of great practical importance, for example, for the design of lubricants. Although the effect has been observed in large-scale atomistic simulations of bulk liquids, its theoretical formulation and microscopic origin are not well understood. Here, we use dissipation-corrected targeted molecular dynamics, which pulls apart two tagged liquid molecules in the presence of surrounding molecules, and analyze this nonequilibrium process via a generalized Langevin equation. The approach is based on a second-order cumulant expansion of Jarzynski's identity, which is shown to be valid for fluids and therefore allows for an exact computation of the friction profile as well of the underlying memory kernel. We show that velocity-dependent friction in fluids results from an intricate interplay of near-order structural effects and the non-Markovian behavior of the friction memory kernel. For complex fluids such as the model lubricant C 40 H 82 , the memory kernel exhibits a stretched-exponential long-time decay, which reflects the multitude of timescales of the system.

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response by integrating quantitative experiments with mathematical modeling and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2α, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 messenger RNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

Ion channels are important proteins for physiological information transfer and functional control. To predict the microscopic origins of their voltage-conductance characteristics, here we applied dissipation-corrected targeted molecular dynamics in combination with Langevin equation simulations to potassium diffusion through the gramicidin A channel as a test system. Performing a nonequilibrium principal component analysis on backbone dihedral angles, we find coupled protein-ion dynamics to occur during ion transfer. The dissipation-corrected free energy profiles correspond well to predictions from other biased simulation methods. The incorporation of an external electric field in Langevin simulations enables the prediction of macroscopic observables in the form of I - V characteristics.

In the rapidly expanding field of peptide therapeutics, the short in vivo half-life of peptides represents a considerable limitation for drug action. D-peptides, consisting entirely of the dextrorotatory enantiomers of naturally occurring levorotatory amino acids (AAs), do not suffer from these shortcomings as they are intrinsically resistant to proteolytic degradation, resulting in a favourable pharmacokinetic profile. To experimentally identify D-peptide binders to interesting therapeutic targets, so-called mirror-image phage display is typically performed, whereby the target is synthesized in D-form and L-peptide binders are screened as in conventional phage display. This technique is extremely powerful, but it requires the synthesis of the target in D-form, which is challenging for large proteins. Here we present finDr, a novel web server for the computational identification and optimization of D-peptide ligands to any protein structure (https://findr.biologie.uni-freiburg.de/). finDr performs molecular docking to virtually screen a library of helical 12-mer peptides extracted from the RCSB Protein Data Bank (PDB) for their ability to bind to the target. In a separate, heuristic approach to search the chemical space of 12-mer peptides, finDr executes a customizable evolutionary algorithm (EA) for the de novo identification or optimization of D-peptide ligands. As a proof of principle, we demonstrate the validity of our approach to predict optimal binders to the pharmacologically relevant target phenol soluble modulin alpha 3 (PSMα3), a toxin of methicillin-resistant Staphylococcus aureus (MRSA). We validate the predictions using in vitro binding assays, supporting the success of this approach. Compared to conventional methods, finDr provides a low cost and easy-to-use alternative for the identification of D-peptide ligands against protein targets of choice without size limitation. We believe finDr will facilitate D-peptide discovery with implications in biotechnology and biomedicine., Competing Interests: None., (© 2021 The Authors.)

Image partitioning, or segmentation without semantics, is the task of decomposing an image into distinct segments, or equivalently to detect closed contours. Most prior work either requires seeds, one per segment; or a threshold; or formulates the task as multicut / correlation clustering, an NP-hard problem. Here, we propose an efficient algorithm for graph partitioning, the "Mutex Watershed". Unlike seeded watershed, the algorithm can accommodate not only attractive but also repulsive cues, allowing it to find a previously unspecified number of segments without the need for explicit seeds or a tunable threshold. We also prove that this simple algorithm solves to global optimality an objective function that is intimately related to the multicut / correlation clustering integer linear programming formulation. The algorithm is deterministic, very simple to implement, and has empirically linearithmic complexity. When presented with short-range attractive and long-range repulsive cues from a deep neural network, the Mutex Watershed gives the best results currently known for the competitive ISBI 2012 EM segmentation benchmark.

Visualizing, tracking and reconstructing cell lineages in developing embryos has been an ongoing effort for well over a century. Recent advances in light microscopy, labelling strategies and computational methods to analyse complex image datasets have enabled detailed investigations into the fates of cells. Combined with powerful new advances in genomics and single-cell transcriptomics, the field of developmental biology is able to describe the formation of the embryo like never before. In this Review, we discuss some of the different strategies and applications to lineage tracing in live-imaging data and outline software methodologies that can be applied to various cell-tracking challenges., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Given nonstationary data from molecular dynamics simulations, a Markovian Langevin model is constructed that aims to reproduce the time evolution of the underlying process. While at equilibrium the free energy landscape is sampled, nonequilibrium processes can be associated with a biased energy landscape, which accounts for finite sampling effects and external driving. When the data-driven Langevin equation (dLE) approach [ Phys . Rev . Lett . 2015 , 115 , 050602] is extended to the modeling of nonequilibrium processes, an efficient way to calculate multidimensional Langevin fields is outlined. The dLE is shown to correctly account for various nonequilibrium processes, including the enforced dissociation of sodium chloride in water, the pressure-jump induced nucleation of a liquid of hard spheres, and the conformational dynamics of a helical peptide sampled from nonstationary short trajectories.

Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the β-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.

Emergence of the novel pathogenic coronavirus SARS-CoV-2 and its rapid pandemic spread presents challenges that demand immediate attention. Here, we describe the development of a semi-quantitative high-content microscopy-based assay for detection of three major classes (IgG, IgA, and IgM) of SARS-CoV-2 specific antibodies in human samples. The possibility to detect antibodies against the entire viral proteome together with a robust semi-automated image analysis workflow resulted in specific, sensitive and unbiased assay that complements the portfolio of SARS-CoV-2 serological assays. Sensitive, specific and quantitative serological assays are urgently needed for a better understanding of humoral immune response against the virus as a basis for developing public health strategies to control viral spread. The procedure described here has been used for clinical studies and provides a general framework for the application of quantitative high-throughput microscopy to rapidly develop serological assays for emerging virus infections., (© 2020 The Authors. BioEssays published by Wiley Periodicals LLC.)

We report on a study that combines advanced fluorescence methods with molecular dynamics (MD) simulations to cover timescales from nanoseconds to milliseconds for a large protein. This allows us to delineate how ATP hydrolysis in a protein causes allosteric changes at a distant protein binding site, using the chaperone Hsp90 as test system. The allosteric process occurs via hierarchical dynamics involving timescales from nano- to milliseconds and length scales from Ångstroms to several nanometers. We find that hydrolysis of one ATP is coupled to a conformational change of Arg380, which in turn passes structural information via the large M-domain α-helix to the whole protein. The resulting structural asymmetry in Hsp90 leads to the collapse of a central folding substrate binding site, causing the formation of a novel collapsed state (closed state B) that we characterise structurally. We presume that similar hierarchical mechanisms are fundamental for information transfer induced by ATP hydrolysis through many other proteins., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

While allostery is of paramount importance for protein regulation, the underlying dynamical process of ligand (un)binding at one site, resulting time evolution of the protein structure, and change of the binding affinity at a remote site are not well understood. Here the ligand-induced conformational transition in a widely studied model system of allostery, the PDZ2 domain, is investigated by transient infrared spectroscopy accompanied by molecular dynamics simulations. To this end, an azobenzene-derived photoswitch is linked to a peptide ligand in a way that its binding affinity to the PDZ2 domain changes upon switching, thus initiating an allosteric transition in the PDZ2 domain protein. The subsequent response of the protein, covering four decades of time, ranging from ∼1 ns to ∼μs, can be rationalized by a remodeling of its rugged free-energy landscape, with very subtle shifts in the populations of a small number of structurally well-defined states. It is proposed that structurally and dynamically driven allostery, often discussed as limiting scenarios of allosteric communication, actually go hand-in-hand, allowing the protein to adapt its free-energy landscape to incoming signals., Competing Interests: The authors declare no competing interest.

Coarse-graining of fully atomistic molecular dynamics simulations is a long-standing goal in order to allow the description of processes occurring on biologically relevant timescales. For example, the prediction of pathways, rates and rate-limiting steps in protein-ligand unbinding is crucial for modern drug discovery. To achieve the enhanced sampling, we perform dissipation-corrected targeted molecular dynamics simulations, which yield free energy and friction profiles of molecular processes under consideration. Subsequently, we use these fields to perform temperature-boosted Langevin simulations which account for the desired kinetics occurring on multisecond timescales and beyond. Adopting the dissociation of solvated sodium chloride, trypsin-benzamidine and Hsp90-inhibitor protein-ligand complexes as test problems, we reproduce rates from molecular dynamics simulation and experiments within a factor of 2-20, and dissociation constants within a factor of 1-4. Analysis of friction profiles reveals that binding and unbinding dynamics are mediated by changes of the surrounding hydration shells in all investigated systems.

Purpose: To date, surgically treated multifragmentary patellar fractures are still associated with high rates of complications, such as i.e. secondary fracture displacement. Osteosynthesis is most frequently performed with screws and cerclages. To increase primary stability, locking plates have been introduced. However, there is still a lack of biomechanical data supporting the superiority of plate fixation compared to screw fixation with cerclages in these cases. The goal of the present study was to conduct biomechanical comparison of these two techniques under dynamic loading conditions., Methods: A standardized 34-C3 fracture was created in eight pairs of human cadaveric knee joints. Following a randomization protocol, they were fixed with a 3.5 mm anterior locking plate (LP) or cannulated screws with anterior tension band wiring (hybrid osteosynthesis, HO).Subsequently, all constructs were tested for 100 cycles from 90° of knee-flexion to full extension by applying a pulling force to the quadriceps tendon. Outcome parameters were fracture displacement after one cycle, after 100 cycles and implant loosening. Failure was defined as fracture displacement > 2 mm., Results: Biomechanical testing showed significantly less fracture displacement following LP compared to HO both after the first (p = 0.042) and after 100 cycles (p = 0.025). The difference in loosening was significant as well (p = 0.017). Following HO, 5/8 constructs failed during cyclic loading. There was no failure in the LP group. In the HO group, loosening correlated with bone mineral density (R = - 0.857) which was not observed in the LP group (R = - 0.429)., Conclusion: Anterior locking plate osteosynthesis of comminuted patellar fractures biomechanically provides better primary stability compared to cannulated screws with anterior tension band wiring., Competing Interests: Declaration of Competing Interest Ferdinand C. Wagner: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Mirjam V. Neumann: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Steffen Wolf: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Alexander Jonaszik: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Kaywan Izadpanah: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Stefan Piatek: This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF) Norbert P. Südkamp: A family member of NP Südkamp is executive partner of the Synmedics KOMM. which is not related to this study. This author, their immediate family, and any research foundation with which they are affiliated did not receive any financial payments or other benefits from any commercial entity related to the subject of this article. (Author's signature see individual PDF), (Copyright © 2020. Published by Elsevier Ltd.)

We here report on nonequilibrium targeted molecular dynamics simulations as a tool for the estimation of protein-ligand unbinding kinetics. Correlating simulations with experimental data from SPR kinetics measurements and X-ray crystallography on two small molecule compound libraries bound to the N-terminal domain of the chaperone Hsp90, we show that the mean nonequilibrium work computed in an ensemble of trajectories of enforced ligand unbinding is a promising predictor for ligand unbinding rates. We furthermore investigate the molecular basis determining unbinding rates within the compound libraries. We propose ligand conformational changes and protein-ligand nonbonded interactions to impact on unbinding rates. Ligands may remain longer at the protein if they exhibit strong electrostatic and/or van der Waals interactions with the target. In the case of ligands with a rigid chemical scaffold that exhibit longer residence times, transient electrostatic interactions with the protein appear to facilitate unbinding. Our results imply that understanding the unbinding pathway and the protein-ligand interactions along this path is crucial for the prediction of small molecule ligands with defined unbinding kinetics.

To facilitate the observation of biomolecular energy transport in real time and with single-residue resolution, recent experiments by Baumann et al. ( Angew. Chem. Int. Ed. 2019 , 58 , 2899 , DOI: 10.1002/anie.201812995 ) have used unnatural amino acids β-(1-azulenyl)alanine (Azu) and azidohomoalanine (Aha) to site-specifically inject and probe vibrational energy in proteins. To aid the interpretation of such experiments, non-equilibrium molecular dynamics simulations of the anisotropic energy flow in proteins TrpZip2 and PDZ3 domains are presented. On this account, an efficient simulation protocol is established that accurately mimics the excitation and probing steps of Azu and Aha. The simulations quantitatively reproduce the experimentally found cooling times of the solvated proteins at room temperature and predict that the cooling slows by a factor 2 below the glass temperature of water. In PDZ3, vibrational energy is shown to travel from the initially excited peptide ligand via a complex network of inter-residue contacts and backbone transport to distal regions of the protein. The supposed connection of these energy transport pathways with pathways of allosteric communication is discussed.

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the α-helical content of the S-peptide reduces upon cis -to- trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the α-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

Principal component analysis (PCA) represents a standard approach to identify collective variables {x i } = x, which can be used to construct the free energy landscape ΔG(x) of a molecular system. While PCA is routinely applied to equilibrium molecular dynamics (MD) simulations, it is less obvious as to how to extend the approach to nonequilibrium simulation techniques. This includes, e.g., the definition of the statistical averages employed in PCA as well as the relation between the equilibrium free energy landscape ΔG(x) and the energy landscapes ΔG(x) obtained from nonequilibrium MD. As an example for a nonequilibrium method, "targeted MD" is considered which employs a moving distance constraint to enforce rare transitions along some biasing coordinate s. The introduced bias can be described by a weighting function P(s), which provides a direct relation between equilibrium and nonequilibrium data, and thus establishes a well-defined way to perform PCA on nonequilibrium data. While the resulting distribution P(x) and energy ΔG∝lnP will not reflect the equilibrium state of the system, the nonequilibrium energy landscape ΔG(x) may directly reveal the molecular reaction mechanism. Applied to targeted MD simulations of the unfolding of decaalanine, for example, a PCA performed on backbone dihedral angles is shown to discriminate several unfolding pathways. Although the formulation is in principle exact, its practical use depends critically on the choice of the biasing coordinate s, which should account for a naturally occurring motion between two well-defined end-states of the system.

As standard unbiased molecular dynamics (MD) simulations become impractical for sampling rare events, "targeted MD" employs a moving distance constraint to enforce rare transitions along some reaction coordinate x. To calculate free energy profiles from these nonequilibrium simulations via Δ G( x) = W( x) - W diss ( x), apart from the (readily obtained) work W( x) performed on the system, the dissipated work W diss ( x) is also required. By employing a second-order cumulant expansion of Jarzynski's equality combined with an analysis within Langevin theory, the dissipated work can be expressed via a nonequilibrium friction coefficient Γ NEQ ( x) that may be calculated on-the-fly from constraint force fluctuations. Adopting the ion dissociation of NaCl in water as a test system, this friction correction is shown to result in accurate free energy profiles, even for a modest number of simulations and at high constraint velocities. As a bonus, the analysis of Γ NEQ ( x) may yield valuable insight into the microscopic mechanism of friction.

The noncanonical amino acid azidohomoalanine (Aha) is known to be an environment-sensitive infrared probe for the site-specific investigation of protein structure and dynamics. Here, the capability of that label is explored to detect protein-ligand interactions by incorporating it in the vicinity of the binding groove of a PDZ2 domain. Circular dichroism and isothermal titration calorimetry measurements reveal that the perturbation of the protein system by mutation is negligible, with minimal influence on protein stability and binding affinity. Two-dimensional infrared spectra exhibit small (1-3 cm -1 ) but clearly measurable red shifts of the Aha vibrational frequency upon binding of two different peptide ligands, while accompanying molecular dynamics simulations suggest that these red shifts are induced by polar contacts with side chains of the peptide ligands. Hence, Aha is a versatile and minimally invasive vibrational label that is not only able to report on large structural changes during, e.g., protein folding, but also on very subtle changes of the electrostatic environment upon ligand binding.

Purpose: The aim of this study was to assess a novel 60-channel receiver body coil (B60) setup for accelerated contrast-enhanced abdominal magnetic resonance imaging (MR) imaging with respect to signal-to-noise ratio (SNR) and image quality using high parallel-imaging technique (PAT) factors in comparison to a standard 30-channel coil setup., Methods: All imaging data were acquired on a 3 T MR scanner using a novel B60 receiver coil setup in comparison to a standard 30-channel setup serving as reference standard. Phantom measurements were performed to systematically evaluate SNR and geometry factor performance in an ex vivo setting. To assess the in vivo application, additional measurements in 5 healthy volunteers and 17 patients were performed using a 3-dimensional T1w gradient-echo sequences with different acceleration factors. At first, the volunteers were examined. In a second step, standard contrast-enhanced abdominal images were acquired in the 17 patients, who were randomly assigned into 2 groups (group A: 60-channel, n = 8; group B: 30-channel, n = 9). Image quality, noise, lesion conspicuity, and artifacts were assessed by 2 radiologists independently on a 5-point Likert scale (5 = excellent)., Results: The phantom study revealed substantial advantages (SNR and geometry factor) of the B60 coil for measurements in the head-to-feet phase encoding direction, whereas only minor differences were found in other directions. Comparison of image quality in volunteers revealed significantly higher ratings of the B60 setup for all acquisitions (P ≤ 0.032), except for PAT = none. In patients, similar results were observed with comparable image quality at lower PAT factors and significantly superior image quality of the B60 setup for higher PAT factors (2 × 2 and 3 × 2; P = 0.008)., Conclusions: The 60-channel coil setup facilitates improved SNR and image quality at high PAT factors with diagnostic image quality, which allows for accelerated contrast-enhanced MR imaging of the abdomen.

We explore the capability of the non-natural amino acid azidohomoalanine (AHA) as an IR label to sense relatively small structural changes in proteins with the help of 2D IR difference spectroscopy. To that end, we AHA-labeled an allosteric protein (the PDZ2 domain from human tyrosine-phosphatase 1E) and furthermore covalently linked it to an azobenzene-derived photoswitch as to mimic its conformational transition upon ligand binding. To determine the strengths and limitations of the AHA label, in total six mutants have been investigated with the label at sites with varying properties. Only one mutant revealed a measurable 2D IR difference signal. In contrast to the commonly observed frequency shifts that report on the degree of solvation, in this case we observe an intensity change. To understand this spectral response, we performed classical MD simulations, evaluating local contacts of the AHA labels to water molecules and protein side chains and calculating the vibrational frequency on the basis of an electrostatic model. Although these simulations revealed in part significant and complex changes of the number of intraprotein and water contacts upon trans-cis photoisomerization, they could not provide a clear explanation of why this one label would stick out. Subsequent quantum-chemistry calculations suggest that the response is the result of an electronic interaction involving charge transfer of the azido group with sulfonate groups from the photoswitch. To the best of our knowledge, such an effect has not been described before.

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.

We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

While adequately chosen reaction coordinates are expected to reveal the mechanism of a dynamical process, it proves to be notoriously difficult to model the complex structural rearrangements of a macromolecule by a low-dimensional collective coordinate. Adopting the hinge-bending motion of T4 lysozyme (T4L) as a prominent example and performing a 50 μs long unbiased molecular dynamics (MD) simulation of T4L, a general strategy to identify reaction coordinates of protein functional dynamics is developed. As a systematic method to reduce the dimensionality of the dynamics, first various types of principal component analyses are employed, and it is shown that the applicability and outcome of the approach crucially depends on the type of input coordinates used. In a second step, prospective candidates for a reaction coordinate are tested by studying the molecule's response to external pulling along the coordinate, using targeted MD simulations. While trying to directly enforce the open-closed transition does not recover the two-state behavior of T4L, this transition is triggered by a locking mechanism, by which the side chain of Phe4 changes from a solvent-exposed to a hydrophobically buried state. The mechanism is found to stabilize the open and closed states of T4L and thereby causes their relatively long lifetime of ∼10 μs. In extension of the usual two-state picture, a four-state model of the functional motion of T4L is proposed, which describes a hierarchical coupling of the fast nanosecond opening-closing motion and the slow microsecond locking transition.

We performed an analysis of possible mechanisms of ligand recognition in the human nose. The analysis is based on in vivo odor threshold determination and in vitro Ca2+ imaging assays with a C/Si/Ge/Sn switch strategy applied to the compounds Lilial and Bourgeonal, to differentiate between different molecular mechanisms of odorant detection. Our results suggest that odorant detection under threshold conditions is mainly based on the molecular shape, i.e. the van der Waals surface, and electrostatics of the odorants. Furthermore, we show that a single olfactory receptor type is responsible for odor detection of Bourgeonal at the threshold level in humans in vivo. Carrying out a QM analysis of vibrational energies contained in the odorants, there is no evidence for a vibration-based recognition.

Background: To evaluate complications and identify risk factors for adverse events in patients undergoing high-dose-rate interstitial brachytherapy (iBT)., Material and Methods: Data from 192 patients treated in 343 CT- or MRI-guided interventions from 2006-2009 at our institution were analyzed. In 41 %, the largest tumor treated was ≥ 5 cm, 6 % of the patients had tumors ≥ 10 cm. Prior to iBT, 60 % of the patients had chemotherapy, 22 % liver resection, 19 % thermoablation or transarterial chemoembolization (TACE). Safety was the primary endpoint; survival data were obtained as the secondary endpoints. During follow-up, MRI or CT imaging was performed and clinical and laboratory parameters were obtained., Results: The rate of major complications was below 5 %. Five major bleedings (1.5 %) occurred. The frequency of severe bleeding was significantly higher in patients with advanced liver cirrhosis. One patient developed signs of a nonclassic radiation-induced liver disease. In 3 patients, symptomatic gastrointestinal (GI) ulcers were detected. A dose exposure to the GI wall above 14 Gy/ml was a reliable threshold to predict ulcer formation. A combination of C-reactive protein ≥ 165 mg/l and/or leukocyte count ≥ 12.7 Gpt/l on the second day after the intervention predicted infection (sensitivity 90.0 %; specificity 92.8 %.) Two patients (0.6 %) died within 30 days. Median overall survival after the first liver treatment was 20.1 months for all patients and the local recurrence-free surviving proportion was 89 % after 12 months., Conclusions: Image-guided iBT yields a low rate of major complications and is effective.

Spherical silver nanoparticles were prepared by means of ion beam synthesis in lithium niobate. The embedded nanoparticles were then irradiated with energetic (84)Kr and (197)Au ions, resulting in different electronic energy losses between 8.1 and 27.5 keV nm(-1) in the top layer of the samples. Due to the high electronic energy losses of the irradiating ions, molten ion tracks are formed inside the lithium niobate in which the elongated Ag nanoparticles are formed. This process is strongly dependent on the initial particle size and leads to a broad aspect ratio distribution. Extinction spectra of the samples feature the extinction maximum with shoulders on either side. While the maximum is caused by numerous remaining spherical nanoparticles, the shoulders can be attributed to elongated particles. The latter could be verified by COMSOL simulations. The extinction spectra are thus a superposition of the spectra of all individual particles.

Schematic presentation of the overall adhesion G Protein-Coupled Receptor (aGPCR) structure and functional domains, covering an extracellular N-terminal fragment (NTF), a membrane-spanning C-terminal fragment (CTF) and a GPCR proteolysis site (GPS). (Left side) aGPCR model constructed based on the seven-transmembrane (7TM) structure (blue) of secretin family glucagon receptor (GCGR) (PDB, 4L6R) [11] and the GPCR autoproteolysis inducing (GAIN) domain (magenta) structure of latrophilin 1 (PDB, 4DLQ) [9]. The β-13 strand residues are depicted in green. (Right side) The experimentally validated full-length secretin family GCGR structure combining structural and experimental information from the GCGR 7TM crystal structure (PDB, 4L6R) (blue), the GCGR extracellular domain (ECD) structure (PDB, 4ERS) (magenta) and the ECD structure of glucagon-like peptide-1 (GLP-1)-bound glucagon-like peptide-1 receptor (GLP-1R) (PDB, 3IOL) (green), complemented by site-directed mutagenesis, electron microscopy (EM), hydrogen-deuterium exchange (HDX) and cross-linking studies [11-13]) Despite the recent breakthroughs in the elucidation of the three-dimensional structures of the seven transmembrane (7TM) domain of the G protein-coupled receptor (GPCR) superfamily, a corresponding structure of a member of the adhesion GPCR (aGPCR) family has not yet been solved. In this chapter, we give an overview of the current knowledge of the 7TM domain of aGPCRs by comparative structure-based sequence similarity analyses between aGPCRs and GPCRs with known crystal structure. Of the GPCR superfamily, only the secretin family shares some sequence similarity with aGPCRs. This chapter will therefore emphasize on the comparison of these two GPCR families. Two 7TM domain structures of secretin family GPCRs are known that provide insight into the structure-function relationships of conserved sequence motifs that play important roles and are also present in most aGPCRs. This suggests that the 7TM domains of aGPCRs and secretin family GPCRs share a similar structural fold and that the conserved residues in both families may be involved in similar intermolecular interaction networks and facilitate similar conformational changes. Comparison of the residues that line the large peptide hormone binding pocket in the 7TM domain of secretin family GPCRs with corresponding residues in aGPCRs indicates that in the latter, the corresponding pocket in the 7TM domain is relatively hydrophobic and may be even larger. Improved knowledge on these conserved sequence motifs will help to understand the interactions of the aGPCR 7TM domain with ligands and gain insight into the activation mechanism of aGPCRs.

Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs.

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.

The findings of Ho et al. in this issue of Science Signaling suggest that the anesthetic ketamine binds to and activates select olfactory receptors in mouse brain, raising the possibility that ketamine targets a similar set of GPCRs in humans., (Copyright © 2015, American Association for the Advancement of Science.)

Proton conduction along protein-bound "water wires" is an essential feature in membrane proteins. Here, we analyze in detail a transient water wire, which conducts protons via a hydrophobic barrier within a membrane protein to create a proton gradient. It is formed only for a millisecond out of three water molecules distributed at inactive positions in a polar environment in the ground state. The movement into a hydrophobic environment causes characteristic shifts of the water bands reflecting their different chemical properties. These band shifts are identified by time-resolved Fourier Transform Infrared difference spectroscopy and analyzed by biomolecular Quantum Mechanical/Molecular Mechanical simulations. A non-hydrogen bonded ("dangling") O-H stretching vibration band and a broad continuum absorbance caused by a combined vibration along the water wire are identified as characteristic marker bands of such water wires in a hydrophobic environment. The results provide a basic understanding of water wires in hydrophobic environments.

The role of protein-bound water molecules in protein function and catalysis is an emerging topic. Here, we studied the solvation of an excess proton by protein-bound water molecules and the contribution of the surrounding amino acid residues at the proton release site of the membrane protein bacteriorhodopsin. It hosts an excess proton within a protein-bound water cluster, which is hydrogen bonded to several surrounding amino acids. Indicative of delocalization is a broad continuum absorbance experimentally observed by time-resolved Fourier transform infrared spectroscopy. In combination with site-directed mutagenesis, the involvement of several amino acids (especially Glu-194 and Glu-204) in the delocalization was elaborated. Details regarding the contributions of the glutamates and water molecules to the delocalization mode in biomolecular simulations are controversial. We carried out quantum mechanics/molecular mechanics (QM/MM) self-consistent charge density functional tight-binding simulations for all amino acids that have been experimentally shown to be involved in solvation of the excess proton, and systematically investigated the influence of the quantum box size. We compared calculated theoretical infrared spectra with experimental ones as a measure for the correct description of excess proton delocalization. A continuum absorbance can only be observed for small quantum boxes containing few amino acids and/or water molecules. Larger quantum boxes, including all experimentally shown involved amino acids, resulted in narrow absorbance bands, indicating protonation of a single binding site in contradiction to experimental results. We conclude that small quantum boxes seem to reproduce representative extreme cases of proton delocalization modes: proton delocalization only on water molecules or only between Glu-194 and Glu-204. Extending the experimental spectral region to lower wave numbers, a water-delocalized proton reproduces the observed continuum absorbance better than a glutamate-shared delocalized proton. However, a full agreement between QM simulations and experimental results on the delocalized excess proton will require a larger quantum box as well as more sophisticated QM/MM methods., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Protein-bound internal water molecules are essential features of the structure and function of microbial rhodopsins. Besides structural stabilization, they act as proton conductors and even proton storage sites. Currently, the most understood model system exhibiting such features is bacteriorhodopsin (bR). During the last 20 years, the importance of water molecules for proton transport has been revealed through this protein. It has been shown that water molecules are as essential as amino acids for proton transport and biological function. In this review, we present an overview of the historical development of this research on bR. We furthermore summarize the recently discovered protein-bound water features associated with proton transport. Specifically, we discuss a pentameric water/amino acid arrangement close to the protonated Schiff base as central proton-binding site, a protonated water cluster as proton storage site at the proton-release site, and a transient linear water chain at the proton uptake site. We highlight how protein conformational changes reposition or reorient internal water molecules, thereby guiding proton transport. Last, we compare the water positions in bR with those in other microbial rhodopsins to elucidate how protein-bound water molecules guide the function of microbial rhodopsins. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks., (© 2013 Elsevier B.V. All rights reserved.)

Silver nanoparticles (NPs) embedded in lithium niobate were fabricated via ion beam synthesis and are suitable for various plasmonic applications, e.g. enhancement of optical nonlinear effects. After room temperature silver implantation, annealing in the temperature range of 400-600 °C was performed in order to recrystallize the damaged lithium niobate surface layer. The shape of the silver NPs, their optical properties as well as the structural properties of their surrounding matrix have been analyzed for various annealing steps. TEM investigations show that annealing at 400 °C does not lead to recrystallization of the damaged lithium niobate. A recrystallization occurs upon increasing the annealing temperature to 500 or 600 °C, but simultaneously a second phase consisting of lithium triniobate forms. This is additionally supported by XRD measurements. By utilizing dynamic annealing, i.e. implanting silver at elevated temperatures of 400 °C, it is shown that the LiNbO3 matrix stays single crystalline during ion implantation and no LiNb3O8 is formed. This is additionally verified by comparing the positions of the surface plasmon resonances with calculations based on Mie's scattering theory.

The light-activated microbial ion channel channelrhodopsin-2 (ChR2) is a powerful tool to study cellular processes with high spatiotemporal resolution in the emerging field of optogenetics. To customize the channel properties for optogenetic experiments, a detailed understanding of its molecular reaction mechanism is essential. Here, Glu-90, a key residue involved in the gating and selectivity mechanism of the ion channel is characterized in detail. The deprotonation of Glu-90 during the photocycle is elucidated by time-resolved FTIR spectroscopy, which seems to be part of the opening mechanism of the conductive pore. Furthermore, Glu-90 is crucial to ion selectivity as also revealed by mutation of this residue combined with voltage clamp experiments. By dynamic homology modeling, we further hypothesized that the conductive pore is flanked by Glu-90 and located between helices A, B, C, and G.