Extending fragment-based free energy calculations with library Monte Carlo simulation: annealing in interaction space

Pre-calculated libraries of molecular fragment configurations have previously been used as a basis for both equilibrium sampling (via library-based Monte Carlo) and for obtaining absolute free energies using a polymer-growth formalism. Here, we combine the two approaches to extend the size of systems for which free energies can be calculated. We study a series of all-atom poly-alanine systems in a simple dielectric solvent and find that precise free energies can be obtained rapidly. For instance, for 12 residues, less than an hour of single-processor time is required. The combined approach is formally equivalent to the annealed importance sampling algorithm; instead of annealing by decreasing temperature, however, interactions among fragments are gradually added as the molecule is grown. We discuss implications for future binding affinity calculations in which a ligand is grown into a binding site.     

Efficient estimation of binding free energies between peptides and an MHC class II molecule using coarse‐grained molecular dynamics simulations with a weighted histogram analysis method

We estimate the binding free energy between peptides and an MHC class II molecule using molecular dynamics (MD) simulations with the weighted histogram analysis method (WHAM). We show that, owing to its more thorough sampling in the available computational time, the binding free energy obtained by pulling the whole peptide using a coarse‐grained (CG) force field (MARTINI) is less prone to significant error induced by inadequate‐sampling than using an atomistic force field (AMBER). We further demonstrate that using CG MD to pull 3–4 residue peptide segments while leaving the remaining peptide segments in the binding groove and adding up the binding free energies of all peptide segments gives robust binding free energy estimations, which are in good agreement with the experimentally measured binding affinities for the peptide sequences studied. Our approach thus provides a promising and computationally efficient way to rapidly and reliably estimate the binding free energy between an arbitrary peptide and an MHC class II molecule. © 2017 Wiley Periodicals, Inc.     

Fragmentation behavior of a thiourea‐based reagent for protein structure analysis by collision‐induced dissociative chemical cross‐linking

The fragmentation behavior of a novel thiourea‐based cross‐linker molecule specifically designed for collision‐induced dissociation (CID) MS/MS experiments is described. The development of this cross‐linker is part of our ongoing efforts to synthesize novel reagents, which create either characteristic fragment ions or indicative constant neutral losses (CNLs) during tandem mass spectrometry allowing a selective and sensitive analysis of cross‐linked products. The new derivatizing reagent for chemical cross‐linking solely contains a thiourea moiety that is flanked by two amine‐reactive N‐hydroxy succinimide (NHS) ester moieties for reaction with lysines or free N‐termini in proteins. The new reagent offers simple synthetic access and easy structural variation of either length or functionalities at both ends. The thiourea moiety exhibits specifically tailored CID fragmentation capabilities—a characteristic CNL of 85 u—ensuring a reliable detection of derivatized peptides by both electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) tandem mass spectrometry and as such possesses a versatile applicability for chemical cross‐linking studies. A detailed examination of the CID behavior of the presented thiourea‐based reagent reveals that slight structural variations of the reagent will be necessary to ensure its comprehensive and efficient application for chemical cross‐linking of proteins. Copyright © 2010 John Wiley & Sons, Ltd.     

Virtual‐system‐coupled adaptive umbrella sampling to compute free‐energy landscape for flexible molecular docking

A novel enhanced conformational sampling method, virtual‐system‐coupled adaptive umbrella sampling (V‐AUS), was proposed to compute 300‐K free‐energy landscape for flexible molecular docking, where a virtual degrees of freedom was introduced to control the sampling. This degree of freedom interacts with the biomolecular system. V‐AUS was applied to complex formation of two disordered amyloid‐β (Aβ_30–35) peptides in a periodic box filled by an explicit solvent. An interpeptide distance was defined as the reaction coordinate, along which sampling was enhanced. A uniform conformational distribution was obtained covering a wide interpeptide distance ranging from the bound to unbound states. The 300‐K free‐energy landscape was characterized by thermodynamically stable basins of antiparallel and parallel β‐sheet complexes and some other complex forms. Helices were frequently observed, when the two peptides contacted loosely or fluctuated freely without interpeptide contacts. We observed that V‐AUS converged to uniform distribution more effectively than conventional AUS sampling did. © 2015 Wiley Periodicals, Inc.     

ss‐NMR and single‐crystal X‐ray diffraction in the elucidation of a new polymorph of bischalcone (1E,4E)‐1,5‐bis(4‐fluorophenyl)penta‐1,4‐dien‐3‐one

We report a new polymorph of (1E,4E)‐1,5‐bis(4‐fluorophenyl)penta‐1,4‐dien‐3‐one, C₁₇H₁₂F₂O. Contrary to the precedent literature polymorph with Z′ = 3, our polymorph has one half molecule in the asymmetric unit disordered over two 50% occupancy sites. Each site corresponds to one conformation around the single bond vicinal to the carbonyl group (so‐called anti or syn). The other half of the bischalcone is generated by twofold rotation symmetry, giving rise to two half‐occupied and overlapping molecules presenting both anti and syn conformations in their open chain. Such a disorder allows for distinct patterns of intermolecular C—H…O contacts involving the carbonyl and anti‐oriented β‐C—H groups, which is reflected in three ¹³C NMR chemical shifts for the carbonyl C atom. Here, we have also assessed the cytotoxicity of three symmetric bischalcones through their in vitro antitumour potential against three cancer cell lines. Cytotoxicity assays revealed that this biological property increases as halogen electronegativity increases.     

The first N‐terminal unprotected (Gly‐Aib)n peptide: H‐Gly‐Aib‐Gly‐Aib‐OtBu

Glycine (Gly) is incorporated in roughly half of all known peptaibiotic (nonribosomally biosynthesized antibiotic peptides of fungal origin) sequences and is the residue with the greatest conformational flexibility. The conformational space of Aib (α‐aminoisobutyric acid) is severely restricted by the second methyl group attached to the C_α atom. Most of the crystal structures containing Aib are N‐terminal protected. Deprotection of the N‐ or C‐terminus of peptides may alter the hydrogen‐bonding scheme and/or the structure and may facilitate crystallization. The structure reported here for glycyl‐α‐aminoisobutyrylglycyl‐α‐aminoisobutyric acid tert‐butyl ester, C₁₆H₃₀N₄O₅, describes the first N‐terminal‐unprotected (Gly‐Aib)_n peptide. The achiral peptide could form an intramolecular hydrogen bond between the C=O group of Gly1 and the N—H group of Aib4. This hydrogen bond is found in all tetrapeptides and N‐terminal‐protected tripeptides containing Aib, apart from one exception. In the present work, this hydrogen bond is not observed (N...O = 5.88 Å). Instead, every molecule is hydrogen bonded to six other symmetry‐related molecules with a total of eight hydrogen bonds per molecule. The backbone conformation starts in the right‐handed helical region (and the left‐handed helical region for the inverted molecule) and reverses the screw sense in the last two residues.     

Real‐Time Monitoring of Phosphorylation Kinetics with Self‐Assembled Nano‐oscillators

Phosphorylation is a post‐translational modification that is involved in many basic cellular processes and diseases, but is difficult to detect in real time with existing technologies. A label‐free detection of phosphorylation is reported in real time with self‐assembled nano‐oscillators. Each nano‐oscillator consists of a gold nanoparticle tethered to a gold surface with a molecular linker. When the nanoparticle is charged, the nano‐oscillator can be driven into oscillation with an electric field and detected with a plasmonic imaging approach. The nano‐oscillators measure charge change associated with phosphorylation of peptides attached onto a single nanoparticle, allowing us to study the dynamic process of phosphorylation in real time without antibodies down to a few molecules, from which Michaelis and catalytic rate constants are determined.     

2‐Ethyl‐1‐[5‐(4‐methylphenyl)pyrazol‐3‐yl]‐3‐(thiophene‐2‐carbonyl)isothiourea: molecular ribbons containing three types of hydrogen‐bonded ring

The title compound, C₁₈H₁₈N₄OS₂, was prepared by reaction of S,S‐diethyl 2‐thenoylimidodithiocarbonate with 5‐amino‐3‐(4‐methylphenyl)‐1H‐pyrazole using microwave irradiation under solvent‐free conditions. In the molecule, the thiophene unit is disordered over two sets of atomic sites, with occupancies of 0.814 (4) and 0.186 (4), and the bonded distances provide evidence for polarization in the acylthiourea fragment and for aromatic type delocalization in the pyrazole ring. An intramolecular N—H...O hydrogen bond is present, forming an S(6) motif, and molecules are linked by N—H...O and N—H...N hydrogen bonds to form a ribbon in which centrosymmetric R₂²(4) rings, built from N—H...O hydrogen bonds and flanked by inversion‐related pairs of S(6) rings, alternate with centrosymmetric R₂²(6) rings built from N—H...N hydrogen bonds.     

Peptide Ligands Stabilized by Small Molecules

Bicyclic peptides generated through directed evolution by using phage display offer an attractive ligand format for the development of therapeutics. Being nearly 100‐fold smaller than antibodies, they promise advantages such as access to chemical synthesis, efficient diffusion into tissues, and needle‐free application. However, unlike antibodies, they do not have a folded structure in solution and thus bind less well. We developed bicyclic peptides with hydrophilic chemical structures at their center to promote noncovalent intramolecular interactions, thereby stabilizing the peptide conformation. The sequences of the peptides isolated by phage display from large combinatorial libraries were strongly influenced by the type of small molecule used in the screen, thus suggesting that the peptides fold around the small molecules. X‐ray structure analysis revealed that the small molecules indeed formed hydrogen bonds with the peptides. These noncovalent interactions stabilize the peptide–protein complexes and contribute to the high binding affinity.     

Development of a Dual‐Functional Hydrogel Using RGD and Anti‐VEGF Aptamer

Synthetic molecular libraries hold great potential to advance the biomaterial development. However, little effort is made to integrate molecules with molecular recognition abilities selected from different libraries into a single biomolecular material. The purpose of this work is to incorporate peptides and nucleic acid aptamers into a porous hydrogel to develop a dual‐functional biomaterial. The data show that an anti‐integrin peptide can promote the attachment and growth of endothelial cells in a 3D porous poly(ethylene glycol) hydrogel and an antivascular endothelial growth factor aptamer can sequester and release VEGF of high bioactivity. Importantly, the dual‐functional porous hydrogel enhances the growth and survival of endothelial cells. This work demonstrates that molecules selected from different synthetic libraries can be integrated into one system for the development of novel biomaterials.     

The Generalized Energy‐Based Fragmentation Approach with an Improved Fragmentation Scheme: Benchmark Results and Illustrative Applications

We propose an improved fragmentation scheme for the generalized energy‐based fragmentation (GEBF) approach, which improves the accuracy of the GEBF approach in total energy calculations and intermolecular interactions. The main modification is to introduce some two‐fragment‐centered primitive subsystems, which are neglected in the previous GEBF implementation. Numerical calculations demonstrate that the present GEBF approach can provide more accurate ground‐state energies and intermolecular interactions. The present GEBF approach with the M06‐2X functional and the cc‐pVTZ basis set are employed to investigate the structures and binding energies in two dimeric species, which are related to pseudopolymorphism of a phenyleneethynylene‐based π‐conjugated molecule. A comparison of the binding free energies in a dimeric species and its corresponding model without C? H???F contacts reveal that the substitution of fluorine atoms weakens the binding of monomers in the dimeric species formed by intermolecular O? H???O hydrogen bonds, but strengthens the binding in the dimer formed by the π–π stacking interaction. Therefore, the C? H???F contacts in these two dimeric species are demonstrated to play a less significant role.     

Coulomb replica‐exchange method: Handling electrostatic attractive and repulsive forces for biomolecules

We propose a new type of the Hamiltonian replica‐exchange method (REM) for molecular dynamics (MD) and Monte Carlo simulations, which we refer to as the Coulomb REM (CREM). In this method, electrostatic charge parameters in the Coulomb interactions are exchanged among replicas while temperatures are exchanged in the usual REM. By varying the atom charges, the CREM overcomes free‐energy barriers and realizes more efficient sampling in the conformational space than the REM. Furthermore, this method requires only a smaller number of replicas because only the atom charges of solute molecules are used as exchanged parameters. We performed Coulomb replica‐exchange MD simulations of an alanine dipeptide in explicit water solvent and compared the results with those of the conventional canonical, replica exchange, and van der Waals REMs. Two force fields of AMBER parm99 and AMBER parm99SB were used. As a result, the CREM sampled all local‐minimum free‐energy states more frequently than the other methods for both force fields. Moreover, the Coulomb, van der Waals, and usual REMs were applied to a fragment of an amyloid‐β peptide (Aβ) in explicit water solvent to compare the sampling efficiency of these methods for a larger system. The CREM sampled structures of the Aβ fragment more efficiently than the other methods. We obtained β‐helix, α‐helix, 3₁₀‐helix, β‐hairpin, and β‐sheet structures as stable structures and deduced pathways of conformational transitions among these structures from a free‐energy landscape. © 2012 Wiley Periodicals, Inc.     

Collective Synthesis of 4‐Hydroxy‐2‐pyridone Alkaloids and Their Antiproliferation Activities

A collective synthesis of 4‐hydroxy‐2‐pyridone alkaloids—specifically, pretenellin B, prebassianin B, farinosone A, militarione D, pyridovericin, and torrubiellone C—has been achieved. Key steps include using a strategic convergent method to synthesize the densely substituted pyridone key intermediate by Suzuki–Miyaura cross‐coupling reaction, a divergent synthesis approach of target molecules by aldol condensation of pyridone intermediate with homologous aldehydes, and an iterative synthesis of homologous aldehydes with all‐trans‐polyene backbones. Interestingly, among the six tumor cell lines investigated, torrubiellone C was found to induce potent and apoptotic inhibitory activities on Jurkat T cells with IC₅₀ values of 7.05 μM . Hence, this approach could potentially contribute to the synthesis of bioactive small‐molecule libraries as well as drug discovery.     

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end‐product was performed by combining proteolytic digestion followed by solid‐phase hydrazide enrichment and liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision‐induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4‐hydroxy‐2‐nonenal (4‐HNE) as a model protein and a representative end‐product of lipid peroxidation, respectively. Although the characteristic elimination of 4‐HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4‐HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid‐phase enrichment with both CID‐ and ECD‐MS/MS are advantageous during an in‐depth interrogation and unequivocal localization of 4‐HNE‐induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues. Copyright © 2010 John Wiley & Sons, Ltd.     

End‐group fidelity in nitroxide‐mediated living free‐radical polymerizations

In this study, new nitroxides based on the 2,2,5‐trimethyl‐4‐phenyl‐3‐azahexane‐3‐oxy skeleton were used to examine chain‐end control during the preparation of polystyrene and poly(t‐butyl acrylate) under living free‐radical conditions. Alkoxyamine‐based initiators with a chromophore attached to either the initiating fragment or the mediating nitroxide fragment were prepared, and the extent of the incorporation of the chromophores at either the initiating end or the propagating chain end was determined. In contrast to 2,2,6,6‐tetramethyl piperidinoxy (TEMPO), the incorporation of the initiating and terminating fragment into the polymer chain was extremely high. For both poly(t‐butyl acrylate) and polystyrene with molecular weights less than or equal to 70,000, incorporations at the initiating end of greater than 97% were observed. At the terminating chain end, incorporations of greater than 95% were obtained for molecular weights less than or equal to 50,000. The level of incorporation tended to decrease slightly at higher molecular weights because of the loss of the alkoxyamine propagating unit, which had important consequences for block copolymer formation. These results clearly show that these new α‐H nitroxides could control the polymerization of vinyl monomers such as styrene and t‐butyl acrylate to an extremely high degree, comparable to anionic and atom transfer radical polymerization procedures. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 4749–4763, 2000     

α‐Peptide–Oligourea Chimeras: Stabilization of Short α‐Helices by Non‐Peptide Helical Foldamers

Short α‐peptides with less than 10 residues generally display a low propensity to nucleate stable helical conformations. While various strategies to stabilize peptide helices have been previously reported, the ability of non‐peptide helical foldamers to stabilize α‐helices when fused to short α‐peptide segments has not been investigated. Towards this end, structural investigations into a series of chimeric oligomers obtained by joining aliphatic oligoureas to the C‐ or N‐termini of α‐peptides are described. All chimeras were found to be fully helical, with as few as 2 (or 3) urea units sufficient to propagate an α‐helical conformation in the fused peptide segment. The remarkable compatibility of α‐peptides with oligoureas described here, along with the simplicity of the approach, highlights the potential of interfacing natural and non‐peptide backbones as a means to further control the behavior of α‐peptides.     

trans,trans‐2‐Cyano‐5‐(4‐methoxy­phenyl)penta‐2,4‐dienethioamide

The title compound, C₁₃H₁₂N₂OS, was prepared by condensation of 4‐methoxycinnamaldehyde and thioacetamide in ethanol at room temperature. Its investigation was undertaken as part of our search for new non‐linear optical compounds. The π‐conjugated title molecule is almost planar, the dihedral angle between the central planar 2‐cyano‐5‐(4‐methoxy­phenyl)penta‐2,4‐diene fragment and the thioacetamide group being 9.2 (6)°. Molecules are linked by N—H?N and N—H?O hydrogen bonds forming a two‐dimensional motif.     

The self‐assembled of cyclic D,L‐α‐peptide systems: Insights into the structure and energetics

Cyclic D,L ‐α‐peptides are able to self‐assemble to nanotubes, although the inherent reason of the stability of this kind of nanotube as well as the intrinsic driving force of self‐assembly of the cyclic D ,L ‐α‐peptides still remain elusive. In this work, using several computational approaches, we investigated the structural and energy characteristics of a series of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] and cyclo[(‐L ‐Ala‐D ‐Ala‐)₄] oligomers. The results reveal that the thermodynamic stability, cooperativity, and self‐assembly patterns of cyclic D ,L ‐α‐peptide nanotubes are mainly determined by the interactions between cross‐strand side chains instead of those between backbones. For cyclo[(‐L ‐Phe‐D ‐Ala‐)₄] oligomers, the steric interaction between cross‐strand side chains, especially the electrostatic repulsion between the phenyls in Phe residues, brings anticooperative effect into parallel stacking mode, which is responsible for the preference of self‐assembling nanotube in antiparallel vs. parallel stacking orientation. Based on our results, a novel self‐assembling mechanism is put forward—it is the L ‐L antiparallel dimer of cyclo[(‐L ‐Phe‐D ‐Ala‐)₄], instead of the commonly presumed monomer, that acts as the basic building block in self assembly. It explains why these cyclic peptides uniquely self‐assemble to form antiparallel nanotubes. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2010     

Direct and Single‐Molecule Visualization of the Solution‐State Structures of G‐Hairpin and G‐Triplex Intermediates

We present the direct and single‐molecule visualization of the in‐pathway intermediates of the G‐quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high‐speed atomic force microscopy (HS‐AFM) for direct visualization, we captured images of the unprecedented solution‐state structures of a tetramolecular antiparallel and (3+1)‐type G‐quadruplex intermediates, such as G‐hairpin and G‐triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid‐state, single‐molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G‐quadruplexes.     

A low‐temperature modification of hexa‐tert‐butyldisilane and a new polymorph of 1,1,2,2‐tetra‐tert‐butyl‐1,2‐diphenyldisilane

Crystals of hexa‐tert‐butyldisilane, C₂₄H₅₄Si₂, undergo a reversible phase transition at 179 (2) K. The space group changes from Ibca (high temperature) to Pbca (low temperature), but the lattice constants a, b and c do not change significantly during the phase transition. The crystallographic twofold axis of the molecule in the high‐temperature phase is replaced by a noncrystallographic twofold axis in the low‐temperature phase. The angle between the two axes is 2.36 (4)°. The centre of the molecule undergoes a translation of 0.123 (1) Å during the phase transition, but the conformation angles of the molecule remain unchanged. Between the two tri‐tert‐butylsilyl subunits there are six short repulsive intramolecular C—H...H—C contacts, with H...H distances between 2.02 and 2.04 Å, resulting in a significant lengthening of the Si—Si and Si—C bonds. The Si—Si bond length is 2.6863 (5) Å and the Si—C bond lengths are between 1.9860 (14) and 1.9933 (14) Å. Torsion angles about the Si—Si and Si—C bonds deviate by approximately 15° from the values expected for staggered conformations due to intramolecular steric H...H repulsions. A new polymorph is reported for the crystal structure of 1,1,2,2‐tetra‐tert‐butyl‐1,2‐diphenyldisilane, C₂₈H₄₆Si₂. It has two independent molecules with rather similar conformations. The Si—Si bond lengths are 2.4869 (8) and 2.4944 (8) Å. The C—Si—Si—C torsion angles deviate by between −3.4 (1) and −18.5 (1)° from the values expected for a staggered conformation. These deviations result from steric interactions. Four Si—C(t‐Bu) bonds are almost staggered, while the other four Si—C(t‐Bu) bonds are intermediate between a staggered and an eclipsed conformation. The latter Si—C(t‐Bu) bonds are about 0.019 (2) Å longer than the staggered Si—C(t‐Bu) bonds.