Publications by authors named "Klaus R Liedl"

181 Publications

Conformational Shifts of Stacked Heteroaromatics: Vacuum vs. Water Studied by Machine Learning.

Front Chem 2021 26;9:641610. Epub 2021 Mar 26.

Center of Molecular Biosciences Innsbruck, Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

Stacking interactions play a crucial role in drug design, as we can find aromatic cores or scaffolds in almost any available small molecule drug. To predict optimal binding geometries and enhance stacking interactions, usually high-level quantum mechanical calculations are performed. These calculations have two major drawbacks: they are very time consuming, and solvation can only be considered using implicit solvation. Therefore, most calculations are performed in vacuum. However, recent studies have revealed a direct correlation between the desolvation penalty, vacuum stacking interactions and binding affinity, making predictions even more difficult. To overcome the drawbacks of quantum mechanical calculations, in this study we use neural networks to perform fast geometry optimizations and molecular dynamics simulations of heteroaromatics stacked with toluene in vacuum and in explicit solvation. We show that the resulting energies in vacuum are in good agreement with high-level quantum mechanical calculations. Furthermore, we show that using explicit solvation substantially influences the favored orientations of heteroaromatic rings thereby emphasizing the necessity to include solvation properties starting from the earliest phases of drug design.
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http://dx.doi.org/10.3389/fchem.2021.641610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032969PMC
March 2021

pyPolyBuilder: Automated Preparation of Molecular Topologies and Initial Configurations for Molecular Dynamics Simulations of Arbitrary Supramolecules.

J Chem Inf Model 2021 Apr 5. Epub 2021 Apr 5.

Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-909, Brazil.

The construction of a molecular topology file is a prerequisite for any classical molecular dynamics simulation. However, the generation of such a file may be very challenging at times, especially for large supramolecules. While many tools are available to provide topologies for large proteins and other biomolecules, the scientific community researching nonbiological systems is not equally well equipped. Here, we present a practical tool to generate topologies for arbitrary supramolecules: The pyPolyBuilder. In addition to linear polymer chains, it also provides the possibility to generate topologies of arbitrary, large, branched molecules, such as, e.g., dendrimers. Furthermore, it also generates reasonable starting structures for simulations of these molecules. pyPolyBuilder is a standalone command-line tool implemented in python. Therefore, it may be easily incorporated in persisting simulation pipelines on any operating systems and with different simulation engines. pyPolyBuilder is freely available on github: https://github.com/mssm-labmmol/pypolybuilder.
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http://dx.doi.org/10.1021/acs.jcim.0c01438DOI Listing
April 2021

Structural determinants of voltage-gating properties in calcium channels.

Elife 2021 Mar 30;10. Epub 2021 Mar 30.

Department of Physiology and Medical Physics, Medical University Innsbruck, Innsbruck, Austria.

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage-sensing properties we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage-sensing domains (VSDs) of the eukaryotic calcium channel Ca1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage-dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.
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http://dx.doi.org/10.7554/eLife.64087DOI Listing
March 2021

Mutation of Framework Residue H71 Results in Different Antibody Paratope States in Solution.

Front Immunol 2021 2;12:630034. Epub 2021 Mar 2.

Department of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innsbruck, Austria.

Characterizing and understanding the antibody binding interface have become a pre-requisite for rational antibody design and engineering. The antigen-binding site is formed by six hypervariable loops, known as the complementarity determining regions (CDRs) and by the relative interdomain orientation (V-V). Antibody CDR loops with a certain sequence have been thought to be limited to a single static canonical conformation determining their binding properties. However, it has been shown that antibodies exist as ensembles of multiple paratope states, which are defined by a characteristic combination of CDR loop conformations and interdomain orientations. In this study, we thermodynamically and kinetically characterize the prominent role of residue 71 (Chothia nomenclature), which does not only codetermine the canonical conformation of the CDR-H2 loop but also results in changes in conformational diversity and population shifts of the CDR-H1 and CDR-H3 loop. As all CDR loop movements are correlated, conformational rearrangements of the heavy chain CDR loops also induce conformational changes in the CDR-L1, CDR-L2, and CDR-L3 loop. These overall conformational changes of the CDR loops also influence the interface angle distributions, consequentially leading to different paratope states in solution. Thus, the type of residue of 71, either an alanine or an arginine, not only influences the CDR-H2 loop ensembles, but co-determines the paratope states in solution. Characterization of the functional consequences of mutations of residue 71 on the paratope states and interface orientations has broad implications in the field of antibody engineering.
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http://dx.doi.org/10.3389/fimmu.2021.630034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960778PMC
March 2021

X-Entropy: A Parallelized Kernel Density Estimator with Automated Bandwidth Selection to Calculate Entropy.

J Chem Inf Model 2021 Mar 13. Epub 2021 Mar 13.

Institute for General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, A-6020 Innsbruck, Austria.

is a Python package used to calculate the entropy of a given distribution, in this case, based on the distribution of dihedral angles. The dihedral entropy facilitates an alignment-independent measure of local protein flexibility. The key feature of our approach is a Gaussian kernel density estimation (KDE) using a plug-in bandwidth selection, which is fully implemented in a C++ backend and parallelized with OpenMP. We further provide a Python frontend, with predefined wrapper functions for classical coordinate-based dihedral entropy calculations, using a 1D approximation. This makes the package very straightforward to include in any Python-based analysis workflow. Furthermore, the frontend allows full access to the C++ backend, so that the KDE can be used on any binnable one-dimensional input data. In this application note, we discuss implementation and usage details and illustrate potential applications. In particular, we benchmark the performance of our module in calculating the entropy of samples drawn from a Gaussian distribution and the analytical solution thereof. Further, we analyze the computational performance of this module compared to well-established python libraries that perform KDE analyses. is available free of charge on GitHub (https://github.com/liedllab/X-Entropy).
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http://dx.doi.org/10.1021/acs.jcim.0c01375DOI Listing
March 2021

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

Brain 2021 Mar 11. Epub 2021 Mar 11.

Institute of Physiology, Medical University Innsbruck, Innsbruck, Austria.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analyzed possible effects on CaV3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with CaV3.3-I860N showing the strongest and CaV3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in CaV3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the CaV3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the CaV3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the CaV3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type CaV3.3 to slow oscillations with CaV3.3-I860N and an intermediate firing mode with CaV3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.
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http://dx.doi.org/10.1093/brain/awab101DOI Listing
March 2021

Inverse relation between structural flexibility and IgE reactivity of Cor a 1 hazelnut allergens.

Sci Rep 2021 Feb 18;11(1):4173. Epub 2021 Feb 18.

Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, 6020, Innsbruck, Austria.

A major proportion of allergic reactions to hazelnuts (Corylus avellana) are caused by immunologic cross-reactivity of IgE antibodies to pathogenesis-related class 10 (PR-10) proteins. Intriguingly, the four known isoforms of the hazelnut PR-10 allergen Cor a 1, denoted as Cor a 1.0401-Cor a 1.0404, share sequence identities exceeding 97% but possess different immunologic properties. In this work we describe the NMR solution structures of these proteins and provide an in-depth study of their biophysical properties. Despite sharing highly similar three-dimensional structures, the four isoforms exhibit remarkable differences regarding structural flexibility, hydrogen bonding and thermal stability. Our experimental data reveal an inverse relation between structural flexibility and IgE-binding in ELISA experiments, with the most flexible isoform having the lowest IgE-binding potential, while the isoform with the most rigid backbone scaffold displays the highest immunologic reactivity. These results point towards a significant entropic contribution to the process of antibody binding.
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http://dx.doi.org/10.1038/s41598-021-83705-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892832PMC
February 2021

pH-Induced Local Unfolding of the Phl p 6 Pollen Allergen From cpH-MD.

Front Mol Biosci 2020 12;7:603644. Epub 2021 Jan 12.

Center for Molecular Biosciences Innsbruck, Institute for General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

Susceptibility to endosomal degradation is a decisive contribution to a protein's immunogenicity. It is assumed that the processing kinetics of structured proteins are inherently linked to their probability of local unfolding. In this study, we quantify the impact of endosomal acidification on the conformational stability of the major timothy grass pollen allergen Phl p 6. We use state of the art sampling approaches in combination with constant pH MD techniques to profile pH-dependent local unfolding events in atomistic detail. Integrating our findings into the current view on type 1 allergic sensitization, we characterize local protein dynamics in the context of proteolytic degradation at neutral and acidic pH for the wild type protein and point mutants with varying proteolytic stability. We analyze extensive simulation data using Markov state models and retrieve highly reliable thermodynamic and kinetic information at varying pH levels. Thereby we capture the impact of endolysosomal acidification on the structure and dynamics of the Phl p 6 mutants. We find that upon protonation at lower pH values, the conformational flexibilities in key areas of the wild type protein, i.e., T-cell epitopes and early proteolytic cleavage sites, increase significantly. A decrease of the pH even leads to local unfolding in otherwise stable secondary structure elements, which is a prerequisite for proteolytic cleavage. This effect is even more pronounced in the destabilized mutant, while no unfolding was observed for the stabilized mutant. In summary, we report detailed structural models which rationalize the experimentally observed cleavage pattern during endosomal acidification.
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http://dx.doi.org/10.3389/fmolb.2020.603644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7835895PMC
January 2021

Surprisingly Fast Interface and Elbow Angle Dynamics of Antigen-Binding Fragments.

Front Mol Biosci 2020 24;7:609088. Epub 2020 Nov 24.

Center for Molecular Biosciences Innsbruck (CMBI), Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

Fab consist of a heavy and light chain and can be subdivided into a variable (V and V ) and a constant region (C 1 and C ). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable loops, shaping the antigen binding site, the paratope. Apart from the CDR loops, both the elbow angle and the relative interdomain orientations of the V -V and the C 1-C domains influence the shape of the paratope. Thus, characterization of the interface and elbow angle dynamics is essential to antigen specificity. We studied nine antigen-binding fragments (Fab) to investigate the influence of affinity maturation, antibody humanization, and different light-chain types on the interface and elbow angle dynamics. While the CDR loops reveal conformational transitions in the micro-to-millisecond timescale, both the interface and elbow angle dynamics occur on the low nanosecond timescale. Upon affinity maturation, we observe a substantial rigidification of the V and V interdomain and elbow-angle flexibility, reflected in a narrower and more distinct distribution. Antibody humanization describes the process of grafting non-human CDR loops onto a representative human framework. As the antibody framework changes upon humanization, we investigated if both the interface and the elbow angle distributions are changed or shifted. The results clearly showed a substantial shift in the relative V -V distributions upon antibody humanization, indicating that different frameworks favor distinct interface orientations. Additionally, the interface and elbow angle dynamics of five antibody fragments with different light-chain types are included, because of their strong differences in elbow angles. For these five examples, we clearly see a high variability and flexibility in both interface and elbow angle dynamics, highlighting the fact that Fab interface orientations and elbow angles interconvert between each other in the low nanosecond timescale. Understanding how the relative interdomain orientations and the elbow angle influence antigen specificity, affinity, and stability has broad implications in the field of antibody modeling and engineering.
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http://dx.doi.org/10.3389/fmolb.2020.609088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732698PMC
November 2020

Conformational Ensembles of Antibodies Determine Their Hydrophobicity.

Biophys J 2021 01 18;120(1):143-157. Epub 2020 Nov 18.

Department of General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria. Electronic address:

A major challenge in the development of antibody biotherapeutics is their tendency to aggregate. One root cause for aggregation is exposure of hydrophobic surface regions to the solvent. Many current techniques predict the relative aggregation propensity of antibodies via precalculated scales for the hydrophobicity or aggregation propensity of single amino acids. However, those scales cannot describe the nonadditive effects of a residue's surrounding on its hydrophobicity. Therefore, they are inherently limited in their ability to describe the impact of subtle differences in molecular structure on the overall hydrophobicity. Here, we introduce a physics-based approach to describe hydrophobicity in terms of the hydration free energy using grid inhomogeneous solvation theory (GIST). We apply this method to assess the effects of starting structures, conformational sampling, and protonation states on the hydrophobicity of antibodies. Our results reveal that high-quality starting structures, i.e., crystal structures, are crucial for the prediction of hydrophobicity and that conformational sampling can compensate errors introduced by the starting structure. On the other hand, sampling of protonation states only leads to good results when combined with high-quality structures, whereas it can even be detrimental otherwise. We conclude by pointing out that a single static homology model may not be adequate for predicting hydrophobicity.
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http://dx.doi.org/10.1016/j.bpj.2020.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820740PMC
January 2021

On the synergy of matrix-isolation infrared spectroscopy and vibrational configuration interaction computations.

Theor Chem Acc 2020 9;139(12):174. Epub 2020 Nov 9.

Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

The key feature of matrix-isolation infrared (MI-IR) spectroscopy is the isolation of single guest molecules in a host system at cryogenic conditions. The matrix mostly hinders rotation of the guest molecule, providing access to pure vibrational features. Vibrational self-consistent field (VSCF) and configuration interaction computations (VCI) on ab initio multimode potential energy surfaces (PES) give rise to anharmonic vibrational spectra. In a single-sourced combination of these experimental and computational approaches, we have established an iterative spectroscopic characterization procedure. The present article reviews the scope of this procedure by highlighting the strengths and limitations based on the examples of water, carbon dioxide, methane, methanol, and fluoroethane. An assessment of setups for the construction of the multimode PES on the example of methanol demonstrates that CCSD(T)-F12 level of theory is preferable to compute (a) accurate vibrational frequencies and (b) equilibrium or vibrationally averaged structural parameters. Our procedure has allowed us to uniquely assign unknown or disputed bands and enabled us to clarify problematic spectral regions that are crowded with combination bands and overtones. Besides spectroscopic assignment, the excellent agreement between theory and experiment paves the way to tackle questions of rather fundamental nature as to whether or not matrix effects are systematic, and it shows the limits of conventional notations used by spectroscopists.
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http://dx.doi.org/10.1007/s00214-020-02682-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7652801PMC
November 2020

Polarizable and non-polarizable force fields: Protein folding, unfolding, and misfolding.

J Chem Phys 2020 Nov;153(18):185102

Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria.

Molecular dynamics simulations are an invaluable tool to characterize the dynamic motions of proteins in atomistic detail. However, the accuracy of models derived from simulations inevitably relies on the quality of the underlying force field. Here, we present an evaluation of current non-polarizable and polarizable force fields (AMBER ff14SB, CHARMM 36m, GROMOS 54A7, and Drude 2013) based on the long-standing biophysical challenge of protein folding. We quantify the thermodynamics and kinetics of the β-hairpin formation using Markov state models of the fast-folding mini-protein CLN025. Furthermore, we study the (partial) folding dynamics of two more complex systems, a villin headpiece variant and a WW domain. Surprisingly, the polarizable force field in our set, Drude 2013, consistently leads to destabilization of the native state, regardless of the secondary structure element present. All non-polarizable force fields, on the other hand, stably characterize the native state ensembles in most cases even when starting from a partially unfolded conformation. Focusing on CLN025, we find that the conformational space captured with AMBER ff14SB and CHARMM 36m is comparable, but the ensembles from CHARMM 36m simulations are clearly shifted toward disordered conformations. While the AMBER ff14SB ensemble overstabilizes the native fold, CHARMM 36m and GROMOS 54A7 ensembles both agree remarkably well with experimental state populations. In addition, GROMOS 54A7 also reproduces experimental folding times most accurately. Our results further indicate an over-stabilization of helical structures with AMBER ff14SB. Nevertheless, the presented investigations strongly imply that reliable (un)folding dynamics of small proteins can be captured in feasible computational time with current additive force fields.
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http://dx.doi.org/10.1063/5.0022135DOI Listing
November 2020

Polyreactive Broadly Neutralizing B cells Are Selected to Provide Defense against Pandemic Threat Influenza Viruses.

Immunity 2020 Dec 22;53(6):1230-1244.e5. Epub 2020 Oct 22.

Department of Medicine, Section of Rheumatology, University of Chicago, Chicago, IL 60637, USA; Committee on Immunology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Polyreactivity is the ability of a single antibody to bind to multiple molecularly distinct antigens and is a common feature of antibodies induced upon pathogen exposure. However, little is known about the role of polyreactivity during anti-influenza virus antibody responses. By analyzing more than 500 monoclonal antibodies (mAbs) derived from B cells induced by numerous influenza virus vaccines and infections, we found mAbs targeting conserved neutralizing influenza virus hemagglutinin epitopes were polyreactive. Polyreactive mAbs were preferentially induced by novel viral exposures due to their broad viral binding breadth. Polyreactivity augmented mAb viral binding strength by increasing antibody flexibility, allowing for adaption to imperfectly conserved epitopes. Lastly, we found affinity-matured polyreactive B cells were typically derived from germline polyreactive B cells that were preferentially selected to participate in B cell responses over time. Together, our data reveal that polyreactivity is a beneficial feature of antibodies targeting conserved epitopes.
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http://dx.doi.org/10.1016/j.immuni.2020.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772752PMC
December 2020

Antibodies exhibit multiple paratope states influencing V-V domain orientations.

Commun Biol 2020 Oct 20;3(1):589. Epub 2020 Oct 20.

Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020, Innsbruck, Austria.

In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as complementarity determining regions (CDRs) shaping the antigen-binding site, the paratope. Whereas it was assumed that certain sequences can only adopt a limited set of backbone conformations, in this study we present a kinetic classification of several paratope states in solution. Using molecular dynamics simulations in combination with experimental structural information we capture the involved conformational transitions between different canonical clusters and additional dominant solution structures occurring in the micro-to-millisecond timescale. Furthermore, we observe a strong correlation of CDR loop movements. Another important aspect when characterizing different paratope states is the relative V/V orientation and the influence of the distinct CDR loop states on the V/V interface. Conformational rearrangements of the CDR loops do not only have an effect on the relative V/V orientations, but also influence in some cases the elbow-angle dynamics and shift the respective distributions. Thus, our results show that antibodies exist as several interconverting paratope states, each contributing to the antibody's properties.
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http://dx.doi.org/10.1038/s42003-020-01319-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7576833PMC
October 2020

Thermosensitive Hydration of Four Acrylamide-Based Polymers in Coil and Globule Conformations.

J Phys Chem B 2020 10 15;124(43):9745-9756. Epub 2020 Oct 15.

Institute of General, Inorganic and Theoretical Chemistry, and Centre of Molecular Biosciences University of Innsbruck, Innsbruck, A-6020 Austria.

To characterize the thermosensitive coil-globule transition in atomistic detail, the conformational dynamics of linear polymer chains of acrylamide-based polymers have been investigated at multiple temperatures. Therefore, molecular dynamic simulations of 30mers of polyacrylamide (AAm), poly--methylacrylamide (NMAAm), poly--ethylacrylamide (NEAAm), and poly--isopropylacrylamide (NIPAAm) have been performed at temperatures ranging from 250 to 360 K for 2 μs. While two of the polymers are known to exhibit thermosensitivity (NEAAm, NIPAAm), no thermosensitivity is observed for AAm and NMAAm in aqueous solution. Our computer simulations consistently reproduce these properties. To understand the thermosensitivity of the respective polymers, the conformational ensembles at different temperatures have been separated according to the coil-globule transition. The coil and globule conformational ensembles were exhaustively analyzed in terms of hydrogen bonding with the solvent, the change of the solvent accessible surface, and enthalpic contributions. Surprisingly, independent of different thermosensitive properties of the four polymers, the surface affinity to water of coil conformations is higher than for globule conformations. Therefore, polymer-solvent interactions stabilize coil conformations at all temperatures. Nevertheless, the enthalpic contributions alone cannot explain the differences in thermosensitivity. This clearly implies that entropy is the distinctive factor for thermosensitivity. With increasing side chain length, the lifetime of the hydrogen bonds between the polymer surface and water is extended. Thus, we surmise that a longer side chain induces a larger entropic penalty due to immobilization of water molecules.
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http://dx.doi.org/10.1021/acs.jpcb.0c07232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604866PMC
October 2020

Design of Phl p 6 Variants With Altered Fold-Stability Significantly Impacts Antigen Processing, Immunogenicity and Immune Polarization.

Front Immunol 2020 18;11:1824. Epub 2020 Aug 18.

Department of Biosciences, University of Salzburg, Salzburg, Austria.

Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. MAESTRO software was used for prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in , and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation , as well as antibody responses and T cell polarization in an adjuvant-free BALB/c mouse model. In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from -5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing . This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. , stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells . This change in processing kinetics significantly influenced the polarization of T cell responses . Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.
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http://dx.doi.org/10.3389/fimmu.2020.01824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7461793PMC
April 2021

-Heterocyclic Carbene Gold(I) Complexes: Mechanism of the Ligand Scrambling Reaction and Their Oxidation to Gold(III) in Aqueous Solutions.

Inorg Chem 2020 Oct 2;59(20):15312-15323. Epub 2020 Oct 2.

Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innrain 80/82, Innsbruck, Tyrol 6020, Austria.

-Heterocyclic carbene (NHC) gold(I) complexes offer great prospects in medicinal chemistry as antiproliferative, anticancer, and antibacterial agents. However, further development requires a thorough understanding of their reaction behavior in aqueous media. Herein, we report the conversion of the bromido[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(I) ((NHC)AuBr, ) complex in acetonitrile/water mixtures to the bis[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(I) ([(NHC)Au], ), which is subsequently oxidized to the dibromidobis[3-ethyl-4-(4-methoxyphenyl)-5-(2-methoxypyridin-5-yl)-1-propylimidazol-2-ylidene]gold(III) ([(NHC)AuBr], ). By combining experimental data from HPLC, NMR, and (LC-)/HR-MS with computational results from DFT calculations, we outline a detailed ligand scrambling reaction mechanism. The key step is the formation of the stacked ((NHC)AuBr) dimer () that rearranges to the T-shaped intermediate Br(NHC)Au-AuBr (). The dissociation of Br from and recombination lead to (NHC)Au-AuBr () followed by the separation into [(NHC)Au] () and [AuBr] (). [AuBr] is not stable in an aqueous environment and degrades in an internal redox reaction to Au and Br. The latter in turn oxidizes to the gold(III) species . The reported ligand rearrangement of the (NHC)AuBr complex differs from that found for related silver(I) analogous. A detailed understanding of this scrambling mechanism is of utmost importance for the interpretation of their biological activity and will help to further optimize them for biomedical and other applications.
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http://dx.doi.org/10.1021/acs.inorgchem.0c02298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7581288PMC
October 2020

Local and Global Rigidification Upon Antibody Affinity Maturation.

Front Mol Biosci 2020 7;7:182. Epub 2020 Aug 7.

Center for Molecular Biosciences Innsbruck, Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

During the affinity maturation process the immune system produces antibodies with higher specificity and activity through various rounds of somatic hypermutations in response to an antigen. Elucidating the affinity maturation process is fundamental in understanding immunity and in the development of biotherapeutics. Therefore, we analyzed 10 pairs of antibody fragments differing in their specificity and in distinct stages of affinity maturation using metadynamics in combination with molecular dynamics (MD) simulations. We investigated differences in flexibility of the CDR-H3 loop and global changes in plasticity upon affinity maturation. Among all antibody pairs we observed a substantial rigidification in flexibility and plasticity reflected in a substantial decrease of conformational diversity. To visualize and characterize these findings we used Markov-states models to reconstruct the kinetics of CDR-H3 loop dynamics and for the first time provide a method to define and localize surface plasticity upon affinity maturation.
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http://dx.doi.org/10.3389/fmolb.2020.00182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426445PMC
August 2020

Decomposing anharmonicity and mode-coupling from matrix effects in the IR spectra of matrix-isolated carbon dioxide and methane.

Phys Chem Chem Phys 2020 Aug;22(32):17932-17947

Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, A-6020 Innsbruck, Austria.

Gas-phase IR spectra of carbon dioxide and methane are nowadays well understood, as a consequence of their pivotal roles in atmospheric- and astrochemistry. However, once those molecules are trapped in noble gas matrices, their spectroscopic properties become difficult to conceptualize. Still, such spectra provide valuable insights into the vibrational structure. In this study, we combine new matrix-isolation infrared (MI-IR) spectra at 6 K in argon and neon with in vacuo anharmonic spectra computed by vibrational self-consistent field (VSCF) and vibrational configuration interaction (VCI). The aim is to separate anharmonicity from matrix effects in the mid-infrared spectra of 12C16O2, 12CH4, and 12CD4. The accurate description of anharmonic potential energy surfaces including mode-coupling allows to reproduce gas-phase data with deviations of below 3 cm-1. Consequently, the remaining difference between MI-IR and VSCF/VCI can be attributed to matrix effects. Frequency shifts and splitting patterns turn out to be unsystematic and dependent on the particular combination of analyte and noble gas. While in the case of neon matrices these effects are small, they are pronounced in xenon, krypton, and argon matrices. Our strategy allows us to suggest that methane rotates in neon matrices - in contrast to previous reports.
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http://dx.doi.org/10.1039/d0cp02121kDOI Listing
August 2020

T-Cell Receptor CDR3 Loop Conformations in Solution Shift the Relative Vα-Vβ Domain Distributions.

Front Immunol 2020 8;11:1440. Epub 2020 Jul 8.

Center for Molecular Biosciences Innsbruck (CMBI), Department of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria.

T-cell receptors are an important part in the adaptive immune system as they are responsible for detecting foreign proteins presented by the major histocompatibility complex (MHC). The affinity is predominantly determined by structure and sequence of the complementarity determining regions (CDRs), of which the CDR3 loops are responsible for peptide recognition. We present a kinetic classification of T-cell receptor CDR3 loops with different loop lengths into canonical and non-canonical solution structures. Using molecular dynamics simulations, we do not only sample available X-ray structures, but we also observe a substantially broader CDR3 loop ensemble with various distinct kinetic minima in solution. Our results strongly imply, that for given CDR3 loop sequences several canonical structures have to be considered to characterize the conformational diversity of these loops. Our suggested dominant solution structures could extend the repertoire of available canonical clusters by including kinetic minimum structures present in solution. Thus, the CDR3 loops need to be characterized as conformational ensembles in solution. Furthermore, the conformational changes of the CDR3 loops follow the paradigm of conformational selection, because the experimentally determined binding competent state is present within this ensemble of pre-existing conformations without the presence of the antigen. We also identify strong correlations between the CDR3 loops and include combined state descriptions. Additionally, we observe a strong dependency of the CDR3 loop conformations on the relative Vα-Vβ interdomain orientations, revealing that certain CDR3 loop states favor specific interface orientations.
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http://dx.doi.org/10.3389/fimmu.2020.01440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360859PMC
July 2020

Conformational selection of allergen-antibody complexes-surface plasticity of paratopes and epitopes.

Protein Eng Des Sel 2019 12;32(11):513-523

Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, A-6020 Innsbruck, Austria.

Antibodies have the ability to bind various types of antigens and to recognize different antibody-binding sites (epitopes) of the same antigen with different binding affinities. Due to the conserved structural framework of antibodies, their specificity to antigens is mainly determined by their antigen-binding site (paratope). Therefore, characterization of epitopes in combination with describing the involved conformational changes of the paratope upon binding is crucial in understanding and predicting antibody-antigen binding. Using molecular dynamics simulations complemented with strong experimental structural information, we investigated the underlying binding mechanism and the resulting local and global surface plasticity in the binding interfaces of distinct antibody-antigen complexes. In all studied allergen-antibody complexes, we clearly observe that experimentally suggested epitopes reveal less plasticity, while non-epitope regions show high surface plasticity. Surprisingly, the paratope shows higher conformational diversity reflected in substantially higher surface plasticity, compared to the epitope. This work allows a visualization and characterization of antibody-antigen interfaces and might have strong implications for antibody-antigen docking and in the area of epitope prediction.
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http://dx.doi.org/10.1093/protein/gzaa014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7451023PMC
December 2019

Profiling selectivity of chagasin mutants towards cysteine proteases cruzain or cathepsin L through molecular dynamics simulations.

J Biomol Struct Dyn 2020 Jul 25:1-13. Epub 2020 Jul 25.

Laboratório de Modelagem Molecular e Planejamento de Fármacos, Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.

Chagasin, an endogenous cysteine protease inhibitor from , can control the activity of the parasitic cruzain and its homologous human cathepsin L. While chagasin inhibits both enzymes with similar potency, mutations have different effects on binding to these enzymes. Mutants T31A and T31A/T32A bind well to cathepsin L, but their affinity for cruzain drops ∼40 to 140-fold. On the other hand, the mutant W93A binds well to cruzain, but it loses potency against cathepsin L. Here, we employed molecular dynamics simulations to understand the selectivity in inhibition of cruzain or cathepsin L by chagasin mutants W93A, T31A, and T31A/T32A. Our results allowed profiling the nonbonded interactions in the interfaces of each mutant with these cysteine proteases. Additionally, we observed differences in the binding conformation of the chagasin loops L2 and L6 of the W93A mutant, favoring interactions with cruzain and reducing interactions with cathepsin L. These differences are associated with a partial dissociation of the W93A-cathepsin L complex, providing a likely cause for the selectivity of the mutant W93A towards cruzain. Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2020.1796797DOI Listing
July 2020

Protein-Protein Binding as a Two-Step Mechanism: Preselection of Encounter Poses during the Binding of BPTI and Trypsin.

Biophys J 2020 08 10;119(3):652-666. Epub 2020 Jul 10.

Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innsbruck, Austria. Electronic address:

Biomolecular recognition between proteins follows complex mechanisms, the understanding of which can substantially advance drug discovery efforts. Here, we track each step of the binding process in atomistic detail with molecular dynamics simulations using trypsin and its inhibitor bovine pancreatic trypsin inhibitor (BPTI) as a model system. We use umbrella sampling to cover a range of unbinding pathways. Starting from these simulations, we subsequently seed classical simulations at different stages of the process and combine them to a Markov state model. We clearly identify three kinetically separated states (an unbound state, an encounter state, and the final complex) and describe the mechanisms that dominate the binding process. From our model, we propose the following sequence of events. The initial formation of the encounter complex is driven by long-range interactions because opposite charges in trypsin and BPTI draw them together. The encounter complex features the prealigned binding partners with binding sites still partially surrounded by solvation shells. Further approaching leads to desolvation and increases the importance of van der Waals interactions. The native binding pose is adopted by maximizing short-range interactions. Thereby side-chain rearrangements ensure optimal shape complementarity. In particular, BPTI's P1 residue adapts to the S1 pocket and prime site residues reorient to optimize interactions. After the paradigm of conformation selection, binding-competent conformations of BPTI and trypsin are already present in the apo ensembles and their probabilities increase during this proposed two-step association process. This detailed characterization of the molecular forces driving the binding process includes numerous aspects that have been discussed as central to the binding of trypsin and BPTI and protein complex formation in general. In this study, we combine all these aspects into one comprehensive model of protein recognition. We thereby contribute to enhance our general understanding of this fundamental mechanism, which is particularly critical as the development of biopharmaceuticals continuously gains significance.
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http://dx.doi.org/10.1016/j.bpj.2020.06.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7399559PMC
August 2020

Replacement of the Cobalt Center of Vitamin B by Nickel: Nibalamin and Nibyric Acid Prepared from Metal-Free B  Ligands Hydrogenobalamin and Hydrogenobyric Acid.

Angew Chem Int Ed Engl 2020 11 2;59(45):20129-20136. Epub 2020 Sep 2.

Institute of Organic Chemistry, University of Innsbruck, 6020, Innsbruck, Austria.

The (formal) replacement of Co in cobalamin (Cbl) by Ni generates nibalamin (Nibl), a new transition-metal analogue of vitamin B . Described here is Nibl, synthesized by incorporation of a Ni ion into the metal-free B  ligand hydrogenobalamin (Hbl), itself prepared from hydrogenobyric acid (Hby). The related Ni  corrin nibyric acid (Niby) was similarly synthesized from Hby, the metal-free cobyric acid ligand. The solution structures of Hbl, and Niby and Nibl, were characterized by spectroscopic studies. Hbl features two inner protons bound at N2 and N4 of the corrin ligand, as discovered in Hby. X-ray analysis of Niby shows the structural adaptation of the corrin ligand to Ni ions and the coordination behavior of Ni . The diamagnetic Niby and Nibl, and corresponding isoelectronic Co corrins, were deduced to be isostructural. Nibl is a structural mimic of four-coordinate base-off Cbls, as verified by its ability to act as a strong inhibitor of bacterial adenosyltransferase.
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http://dx.doi.org/10.1002/anie.202008407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693184PMC
November 2020

Solvation Thermodynamics in Different Solvents: Water-Chloroform Partition Coefficients from Grid Inhomogeneous Solvation Theory.

J Chem Inf Model 2020 08 20;60(8):3843-3853. Epub 2020 Jul 20.

Institute of General, Inorganic and Theoretical Chemistry, and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.

Reliable information on partition coefficients plays a key role in drug development, as solubility decisively affects bioavailability. In a physicochemical context, the partition coefficient of a solute between two different solvents can be described as a function of solvation free energies. Hence, substantial scientific efforts have been made toward accurate predictions of solvation free energies in various solvents. The grid inhomogeneous solvation theory (GIST) facilitates the calculation of solvation free energies. In this study, we introduce an extended version of the GIST algorithm, which enables the calculation for chloroform in addition to water. Furthermore, GIST allows localization of enthalpic and entropic contributions. We test our approach by calculating partition coefficients between water and chloroform for a set of eight small molecules. We report a Pearson correlation coefficient of 0.96 between experimentally determined and calculated partition coefficients. The capability to reliably predict partition coefficients between water and chloroform and the possibility to localize their contributions allow the optimization of a compound's partition coefficient. Therefore, we presume that this methodology will be of great benefit for the efficient development of pharmaceuticals.
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http://dx.doi.org/10.1021/acs.jcim.0c00289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460078PMC
August 2020

Ni[B (SO ) ] and Co[B (SO ) ]: Unveiling Systematic Trends in Phyllosilicate Analogue Borosulfates.

Chemistry 2020 Dec 22;26(72):17405-17415. Epub 2020 Oct 22.

Institute of Inorganic Chemistry, University of Cologne, Greinstrasse 6, 50939, Cologne, Germany.

Borosulfates are compounds analogous to silicates, with heteropolyanionic subunits of vertex-linked (SO )- and (BO )-tetrahedra. In contrast to the immense structural diversity of silicates, the number of borosulfates is yet very limited and the extent of their properties is still unknown. This is particularly true for representatives with phyllosilicate and tectosilicate analogue anionic substructures. Herein, we present Ni[B (SO ) ] and Co[B (SO ) ], two new borosulfates with phyllosilicate analogue topology. While the anionic subunits of both structures are homeotypic, the positions of the charge compensating cations differ significantly: Ni is located between the borosulfate layers, while Co -in contrast-is embedded within the layer. Detailed analysis of these two structures based on single-crystal X-ray diffraction, magnetochemical investigations, X-ray photoelectron spectroscopy, and quantum chemical calculations, unveiled the reasons for this finding. By in silico comparison with other divalent borosulfates, we uncovered systematic trends for phyllosilicate analogues leading to the prediction of new species.
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http://dx.doi.org/10.1002/chem.202002221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820960PMC
December 2020

Macrocycle Cell Permeability Measured by Solvation Free Energies in Polar and Apolar Environments.

J Chem Inf Model 2020 07 29;60(7):3508-3517. Epub 2020 Jun 29.

Institute of General, Inorganic and Theoretical Chemistry and Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80-82, Innsbruck A-6020 Austria.

The relation of surface polarity and conformational preferences is decisive for cell permeability and thus bioavailability of macrocyclic drugs. Here, we employ grid inhomogeneous solvation theory (GIST) to calculate solvation free energies for a series of six macrocycles in water and chloroform as a measure of passive membrane permeability. We perform accelerated molecular dynamics simulations to capture a diverse structural ensemble in water and chloroform, allowing for a direct profiling of solvent-dependent conformational preferences. Subsequent GIST calculations facilitate a quantitative measure of solvent preference in the form of a transfer free energy, calculated from the ensemble-averaged solvation free energies in water and chloroform. Hence, the proposed method considers how the conformational diversity of macrocycles in polar and apolar solvents translates into transfer free energies. Following this strategy, we find a striking correlation of 0.92 between experimentally determined cell permeabilities and calculated transfer free energies. For the studied model systems, we find that the transfer free energy exceeds the purely water-based solvation free energies as a reliable estimate of cell permeability and that conformational sampling is imperative for a physically meaningful model. We thus recommend this purely physics-based approach as a computational tool to assess cell permeabilities of macrocyclic drug candidates.
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http://dx.doi.org/10.1021/acs.jcim.0c00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388155PMC
July 2020

The intermolecular anthracene-transfer in a regiospecific antipodal C difunctionalization.

Org Biomol Chem 2020 Jun 19;18(21):4090-4103. Epub 2020 May 19.

Institute of General, Inorganic and Theoretical Chemistry, and Centre of Molecular Biosciences, University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.

Ever since the discovery of fullerenes, their mono- and multi-functionalization by exohedral addition chemistry has been a fundamental topic. A few years ago, a topochemically controlled regiospecific difunctionalization of C fullerene by anthracene in the solid state was discovered. In the present work, we analyse the mechanism of this unique reaction, where an anthracene molecule is transferred from one C mono-adduct to another one, under exclusive formation of equal amounts of C and of the difficult to make, highly useful, antipodal C bis-adduct. Our herein disclosed dispersion corrected DFT studies show the anthracene transfer to take place in a synchronous retro Diels-Alder/Diels-Alder reaction: an anthracene molecule dissociates from one fullerene under formation of an intermediate, while undergoing stabilizing interactions with both neighbouring fullerene molecules, facilitating the reaction kinetically. In the intermediate, a planar anthracene molecule is sandwiched between two neighbouring fullerenes and forms equally strong 'double-decker' type π-π stacking interactions with both of these fullerenes. Analysis with the distortion interaction model shows that the anthracene unit of the intermediate is almost planar with minimal distortion. This analysis highlights the existence of simultaneous noncovalent interactions engaging both faces of a planar polyunsaturated ring and two convex fullerene surfaces in an unprecedented 'inverted sandwich' structure. Hence, it sheds light on new strategies to design functional fullerene based materials.
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http://dx.doi.org/10.1039/d0ob00520gDOI Listing
June 2020

Charge Anisotropy of Nitrogen: Where Chemical Intuition Fails.

J Chem Theory Comput 2020 Jul 9;16(7):4443-4453. Epub 2020 Jun 9.

Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 80-82, A-6020 Innsbruck, Austria.

For more than half a century computer simulations were developed and employed to study ensemble properties of a wide variety of atomic and molecular systems with tremendous success. Nowadays, a selection of force-fields is available that describe the interactions in such systems. A key feature of force-fields is an adequate description of the electrostatic potential (ESP). Several force-fields model the ESP via point charges positioned at the atom centers. A major shortcoming of this approach, its inability to model anisotropies in the ESP, can be mitigated using additional charge sites. It has been shown that nitrogen is the most problematic element abundant in many polymers as well as large molecules of biological origin. To tackle this issue, small organic molecules containing a single nitrogen atom were studied. In performing rigorous scans of the surroundings of these nitrogen atoms, positions where a single extra charge can enhance the ESP description the most were identified. Significant improvements are found for ammonia, amines, and amides. Interestingly, the optimal location for the extra charge does not correlate with the chemically intuitive position of the nitrogen lone pair. In fact, the placement of an extra charge in the lone-pair location does not lead to significant improvements in most cases.
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http://dx.doi.org/10.1021/acs.jctc.0c00204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365557PMC
July 2020

Catalytic Site p Values of Aspartic, Cysteine, and Serine Proteases: Constant pH MD Simulations.

J Chem Inf Model 2020 06 29;60(6):3030-3042. Epub 2020 May 29.

Institute for General, Inorganic and Theoretical Chemistry, Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria.

Enzymatic function and activity of proteases is closely controlled by the pH value. The protonation states of titratable residues in the active site react to changes in the pH value, according to their p, and thereby determine the functionality of the enzyme. Knowledge of the titration behavior of these residues is crucial for the development of drugs targeting the active site residues. However, experimental p data are scarce, since the systems' size and complexity make determination of these p values inherently difficult. In this study, we use single pH constant pH MD simulations as a fast and robust tool to estimate the active site p values of a set of aspartic, cysteine, and serine proteases. We capture characteristic p shifts of the active site residues, which dictate the experimentally determined activity profiles of the respective protease family. We find clear differences of active site p values within the respective families, which closely match the experimentally determined pH preferences of the respective proteases. These shifts are caused by a distinct network of electrostatic interactions characteristic for each protease family. While we find convincing agreement with experimental data for serine and aspartic proteases, we observe clear deficiencies in the description of the titration behavior of cysteines within the constant pH MD framework and highlight opportunities for improvement. Consequently, with this work, we provide a concise set of active site p values of aspartic and serine proteases, which could serve as reference for future theoretical as well as experimental studies.
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http://dx.doi.org/10.1021/acs.jcim.0c00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312390PMC
June 2020