Carboxylate ion pairing with alkali-metal ions for β-Lactoglobulin and its role on aggregation and interfacial adsorption

We report a combined experimental and computational study of the whey protein β-lactoglobulin (BLG) in different electrolyte solutions. Vibrational sum-frequency generation (SFG) and ellipsometry were used to investigate the molecular structure of BLG modified air–water interfaces as a function of LiCl, NaCl, and KCl concentrations. Molecular dynamics (MD) simulations and thermodynamic integration provided details of the ion pairing of protein surface residues with alkali-metal cations. Our results at pH 6.2 indicate that BLG at the air–water interface forms mono- and bilayers preferably at low and high ionic strength, respectively. Results from SFG spectroscopy and ellipsometry are consistent with intimate ion pairing of alkali-metal cations with aspartate and glutamate carboxylates, which is shown to be more effective for smaller cations (Li⁺ and Na⁺). MD simulations show not only carboxylate–alkali-metal ion pairs but also ion multiplets with the alkali-metal ion in a bridging position between two or more carboxylates. Consequently, alkali-metal cations can bridge carboxylates not only within a monomer but also between monomers, thus providing an important dimerization mechanism between hydrophilic surface patches

2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis

Funding: Engineering and Physical Sciences Research Council [EP/M019195/1]; Engineering and Physical Sciences Research Council Studentship (to J.S.H.); Biotechnology and Biological Sciences Research Council [BB/J001694/2, BB/R021848/1]; ADTBio; University of Kentucky and NCI Cancer Center Support Grant [P30 CA177558]; The Carmen L. Buck Endowment; Emerging Fields Initiative of the Friedrich-Alexander-University of Erlangen-Nuremberg [Grant title ‘Chemistry in Live Cells’]; Wellcome Trust [099149/Z/12/Z]; Royal Society, University Research Fellowship (to J.E.L.). Funding for open access charge: University of Oxford.The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.Publisher PDFPeer reviewe

Tautomeric equilibria of nucleobases in the hachimoji expanded genetic alphabet

Evolution has yielded biopolymers that are constructed from exactly four building blocks and are able to support Darwinian evolution. Synthetic biology aims to extend this alphabet, and we recently showed that 8-letter (hachimoji) DNA can support rule-based information encoding. One source of replicative error in non-natural DNA-like systems, however, is the occurrence of alternative tautomeric forms, which pair differently. Unfortunately, little is known about how structural modifications impact free-energy differences between tautomers of the non-natural nucleo¬bases used in the hachimoji expanded genetic alphabet. Determining experimental tautomer ratios is technically difficult and so strategies for improving hachimoji DNA replication efficiency will benefit from accurate computational predictions of equilibrium tautomeric ratios. We now report that high-level quantum-chemical calculations in aqueous solution by the embedded cluster reference interaction site model (EC-RISM), benchmarked against free energy molecular simulations for solvation thermodynamics, provide useful quantitative information on the tautomer ratios of both Watson-Crick and hachimoji nucleobases. In agreement with previous computational studies, all four Watson-Crick nucleobases adopt essentially only one tautomer in water. This is not the case, however, for non-natural nucleobases and their analogs. For example, although the enols of isoguanine and a series of related purines are not populated in water, these heterocycles possess N1-H and N3-H keto tautomers that are similar in energy thereby adversely impacting accurate nucleobase pairing. These robust computational strategies offer a firm basis for improving experimental measurements of tautomeric ratios, which are currently limited to studying molecules that exist only as two tautomers in solution

Interaction of Thymine DNA Glycosylase with Oxidised 5-Methyl-cytosines in Their Amino- and Imino-Forms

Thymine DNA Glycosylase (TDG) is an enzyme of the base excision repair mechanism and removes damaged or mispaired bases from DNA via hydrolysis of the glycosidic bond. Specificity is of high importance for such a glycosylase, so as to avoid the damage of intact DNA. Among the substrates reported for TDG are mispaired uracil and thymine but also formyl-cytosine and carboxyl-cytosine. Methyl-cytosine and hydroxylmethyl-cytosine are, in contrast, not processed by the TDG enzyme. We have in this work employed molecular dynamics simulations to explore the conformational dynamics of DNA carrying a formyl-cytosine or carboxyl-cytosine and compared those to DNA with the non-cognate bases methyl-cytosine and hydroxylmethyl-cytosine, as amino and imino tautomers. Whereas for the mispairs a wobble conformation is likely decisive for recognition, all amino tautomers of formyl-cytosine and carboxyl-cytosine exhibit the same Watson–Crick conformation, but all imino tautomers indeed form wobble pairs. The conformational dynamics of the amino tautomers in free DNA do not exhibit differences that could be exploited for recognition, and also complexation to the TDG enzyme does not induce any alteration that would indicate preferable binding to one or the other oxidised methyl-cytosine. The imino tautomers, in contrast, undergo a shift in the equilibrium between a closed and a more open, partially flipped state, towards the more open form upon complexation to the TDG enzyme. This stabilisation of the more open conformation is most pronounced for the non-cognate bases methyl-cytosine and hydroxyl-cytosine and is thus not a likely mode for recognition. Moreover, calculated binding affinities for the different forms indicate the imino forms to be less likely in the complexed DNA. These findings, together with the low probability of imino tautomers in free DNA and the indifference of the complexed amino tautomers, suggest that discrimination of the oxidised methyl-cytosines does not take place in the initial complex formation

QM/MM Docking und Simulationen zum Fluoreszenz-Resonanz-Energie-Transfer

The following presents two approaches that use combined quantum mechanical/molecular mechanical (QM/MM) methods in order to gain deeper insights into the structure and function of protein-ligand complexes and to understand complex results of protein spectroscopy by extensive computer simulations. In the first part, a combined QM/MM docking approach for investigating protein-inhibitor complexes is presented. Starting points for QM/MM optimizations are generated with AutoDock. The subsequent semiempirical AM1 QM/MM optimization of the complex obtained by the docking procedure gives a more detailed description of the binding mode and the electronic properties of the ligand. As a flexible protein environment is used in the QM/MM optimizations, the method is able to simulate limited structural changes of the enzyme upon binding a ligand, even within a simple geometry optimization. The method was validated using a set of structurally known protein-inhibitor complexes, whose crystallographic data were taken from the Protein Data Bank. In addition to protein structures taken directly from complexes with the inhibitors, structures of uncomplexed HIV-1-protease and thrombin were also used successfully for QM/MM docking experiments. By comparing the resulting structures with those obtained using protein structures from protein-inhibitor complexes, it is shown that the method is able to simulate the effect of the induced fit when a simple optimization is adequate to reproduce the protein movement. Describing the ligand quantum mechanically gives a detailed view of its electronic properties, e.g. its polarization within the active site of the enzyme. This study suggests strongly that a QM/MM molecular dynamics approach will be able to simulate the induced fit in general cases. A computational model study designed to simulate the results of time-resolved fluorescence spectra of tryptophan in proteins is presented in the second part. In such measurements, the occurrence of more than one fluorescence lifetime is generally attributed to the existence of several tryptophan rotamers and/or structural conformations of the protein structure. The system chosen for this initial study is the tetracycline repressor (TetR) protein, an interesting model system for the investigation of the mechanisms of transcriptional regulation. Fluorescence resonance energy transfer (FRET) from tryptophan to tetracycline is frequently observed in complexes of the Tet-repressor with the antibiotic tetracycline. A combined classical/quantum mechanical approach is used to model the structure and the spectroscopic properties of the TetR-tetracycline complex. A classical molecular dynamics simulation provides input geometries for semiempirical QM/MM CI calculations, which are used to calculate tryptophan absorption and fluorescence spectra as well as fluorescence resonance energy transfer (FRET) rate constants. It is shown how a biexponential tryptophan fluorescence decay can be obtained from a molecular dynamics (MD) trajectory containing two tryptophan rotamers by calculating the FRET rate constant for every MD snapshot. The results indicate that the classical "rotamer model", used to explain the multiexponentiality of time-resolved tryptophan emission spectra, can be extended to systems with FRET acceptors present in the protein matrix.In dieser Arbeit werden zwei neue Ansätze aus der Computerchemie vorgestellt, in denen gemischt quantenmechanische/molekülmechanische (QM/MM) Verfahren verwendet werden, um tiefere Einblicke in die Struktur und die Funktion von Protein-Ligand-Komplexen zu erhalten. Außerdem wird gezeigt, wie umfangreiche Computersimulationen zur Erklärung komplexer Befunde aus der Proteinspektroskopie verwendet werden können. Im ersten Teil der Arbeit wird ein kombinierter QM/MM-Docking-Ansatz vorgestellt, mit dem Protein-Inhibitor-Komplexe untersucht werden. Mit Hilfe des Docking-Programms AutoDock wird die Bindung kleiner organischer Moleküle in der Bindetasche von Enzymen vorhergesagt. Hierbei wird der Ligand als flexibles Molekül behandelt, das Protein ist jedoch starr und es kann keine konformative Anpassung des Proteins (induced fit) als Reaktion auf die Ligandbindung erfolgen. Die durch das Docking erhaltenen Strukturen werden mit semiempirischen AM1 QM/MM-Optimierungen verfeinert. Hierbei wird eine verbesserte Beschreibung des Bindungsmodus des Liganden erhalten. Außerdem liefert die quantenmechanische Rechnung eine korrekte Beschreibung der elektronischen Eigenschaften des Liganden. Durch die Verwendung einer flexiblen Proteinumgebung bei den QM/MM-Rechnungen können strukturelle Anpassungen des Enzyms als Reaktion auf die Ligandbindung (induced fit) sogar im Rahmen einer einfachen Geometrieoptimierung simuliert werden, und auf den Einsatz teuerer Methoden (z.B. Moleküldynamik) kann verzichtet werden. Die Methode wurde mit einem Satz durch Röntgenkristallographie aufgeklärter Protein-Inhibitor-Komplexe validiert, deren Geometrien aus der Proteindatenbank (PDB) entnommen wurden. Hierbei wurden auch Metalloproteine, für die das Dockingprogramm keine Parameter hat, erfolgreich gedockt. Hierzu wurden die Standard-Kraftfeldparameter um geeignete Metallparameter ergänzt. Zusätzlich zu den Strukturen von zusammen mit dem Inhibitor kristallisierten Proteinen wurden auch ligandfrei kristallisierte Strukturen von HIV-1-Protease und Thrombin erfolgreich für Docking-Experimente verwendet. Durch den Vergleich der erhaltenen Strukturen mit den Ergebnissen, die unter Verwendung von mit einem Liganden kristallisierten Proteinstrukturen erhalten wurden, konnte gezeigt werden, daß die Methode begrenzte konformative Anpassungen eines Enzyms nach Bindung eines Liganden auch durch einfache Geometrieoptimierungen simulieren kann und in diesen Fällen auf teurere, rechenzeitintensivere Verfahren verzichtet werden kann. Die quantenmechanische Beschreibung des Liganden liefert ein detailliertes Bild seiner elektronischen Eigenschaften, z. B. seiner Polarisation durch die Gruppen im aktiven Zentrum eines Enzyms. Die Ergebnisse der vorgestellten Arbeit weisen darauf hin, daß ein QM/MM-Moleküldynamik-Ansatz den Effekt des induced fit auch in solchen Fällen simulieren kann, in denen größere konformative Anpassungen des Enzyms, wie z. B. das Öffnen einer Bindetasche, erforderlich sind. Im zweiten Teil der Arbeit wird eine Studie vorgestellt, mit Hilfe derer die Ergebnisse zeitaufgelöster Fluoreszenzspektroskopie der Aminosäure Tryptophan in Proteinen erklärt werden können. Bei derartigen Messungen wird häufig mehr als eine Lebensdauer beobachtet, was gewöhnlich mit der Existenz mehrerer Seitenkettenkonformationen von Tryptophan oder mehrerer Proteinkonformationen erklärt wird ("Rotamerenmodell"). In dieser Arbeit wird das aus zwei Monomeren bestehende Tet-Repressor (TetR)-Protein untersucht, welches ein interessantes Modellsystem für die Erforschung der Mechanismen der transkriptionellen Regulation darstellt. In Komplexen des Tet-Repressors mit Tetracyclin wird häufig Fluoreszenz-Resonanz-Energie-Transfer (Förster-Energie-Transfer, FRET) von der Aminosäure Tryptophan zum Induktor Tetracyclin beobachtet. Die Struktur und Dynamik sowie die spektroskopischen Eigenschaften des TetR-Tetracyclin-Komplexes werden mit Hilfe eines gemischt klassischen/quantenmechanischen Verfahrens untersucht. Eine klassische Moleküldynamik (MD)-Simulation liefert die Eingabegeometrien für semiempirische QM/MM CI-Rechnungen, die verwendet werden, um die zur Simulation der spektroskopischen Eigenschaften der relevanten Chromophore (Tryptophan und Tetracyclin) erforderlichen Größen zu berechnen. Die Analyse der klassischen MD-Simulationen ergab für den Chromophor Tryptophan 43, einer in den DNA-Bindeköpfen der beiden Monomere des TetR-Proteins vorkommenden Aminosäure, zwei (Monomer 1) bzw. fünf (Monomer 2) Rotamere. Zusätzlich zu den Änderungen der Seitenkettenwinkel, die die Rotamere ineinander überführen, wurde eine schnelle Bewegung der Tryptophan-Seitenkette beobachtet. Für die weitere Analyse wurde das Monomer 1, das zwei Rotamere in der MD aufweist, verwendet. Die vorgestellte Methode ist im Gegensatz zu den meisten experimentellen Verfahren in der Lage, die Rotamere unabhängig voneinander zu untersuchen. Neben den stationären Absorptions- und Fluoreszenzspektren von Tryptophan und Tetracyclin wurden auch zeitaufgelöste Fluoreszenzspektren von Tryptophan simuliert. Hierbei wurden zum einen intrinsische (ohne Löschung) Fluoreszenzabklingkurven von Tryptophan aus den Einstein-Koeffizienten, die für jeden MD-Snapshot errechnet wurden, generiert. Die Simulationen zeigen sowohl für die gesamte MD-Trajektorie (2 Rotamere) als auch für die einzelnen Rotamere ein biexponentielles Abklingverhalten. Dieses wird durch die schnelle Bewegung der Tryptophan-Seitenkette, die im Laufe der Simulation zu einer Vielfalt von Tryptophan-Mikroumgebungen führt, verursacht. Zusätzlich zur intrinsischen Fluoreszenz von Tryptophan 43 wurde auch dessen Fluoreszenzlöschung durch Förster-Energietransfer (FRET) zum Induktor Tetracyclin untersucht. Hierzu wurde für jeden MD-Snapshot die Geschwindigkeitskonstante für den Fluoreszenz-Resonanz-Energie-Transfer (FRET) berechnet. Es konnte gezeigt werden, daß sowohl die gesamte MD-Trajektorie (2 Rotamere) als auch die einzelnen Rotamere ein biexponentielles Abklingverhalten, verursacht durch FRET, aufweisen. Die schnellen Bewegungen der Tryptophan-Seitenkettenwinkel führen auch hier zu einer Vielfalt an relativen Donor-/Akzeptorgeometrien, was aufgrund der großen Empfindlichkeit von FRET für derartige Geometrieänderungen das biexponentielle Verhalten verursacht. Die Analyse der relativen FRET-Geschwindigkeitskonstanten zeigt, daß die beiden Rotamere eine unterschiedliche Wahrscheinlichkeit für eine Löschung durch Energietransfer aufweisen. Entsprechend werden für die beiden Rotamere unterschiedliche Lebensdauern gefunden. Diese Ergebnisse zeigen, daß das klassische "Rotamerenmodell", welches gewöhnlich zur Interpretation zeitaufgelöster Emissionsspektren von Tryptophan dient, die tatsächlichen Verhältnisse zu stark vereinfacht, indem es annimmt, daß jedes Rotamer einer diskreten Lebensdauer zuzuordnen ist. Die vorgestellten Ergebnisse zeigen, daß das biexponentielle Abklingverhalten zumindest im Fall des untersuchten TetR/Tetracyclin-Systems aus den schnellen Bewegungen des Tryptophan-Chromophors und den resultierenden mannigfaltigen Donor-/Akzeptorgeometrien folgt. Die unterschiedlichen FRET-Geschwindigkeitskonstanten/Lebensdauern der Rotamere zeigen aber, daß das Rotamerenmodell für Systeme mit FRET-Akzeptoren erweitert werden kann

A simple QM/MM approach for capturing polarization effects in protein-ligand binding free energy calculations

We present a molecular simulation protocol to compute free energies of binding, which combines a QM/MM correction term with rigorous classical free energy techniques, thereby accounting for electronic polarization effects. Relative free energies of binding are first computed using classical force fields, Monte Carlo sampling, and replica exchange thermodynamic integration. Snapshots of the configurations at the end points of the perturbation are then subjected to DFT-QM/MM single-point calculations using the B3LYP functional and a range of basis sets. The resulting quantum mechanical energies are then processed using the Zwanzig equation to give free energies incorporating electronic polarization. Our approach is conceptually simple and does not require tightly coupled QM and MM software. The method has been validated by calculating the relative free energies of hydration of methane and water and the relative free energy of binding of two inhibitors of cyclooxygenase-2. Closed thermodynamic cycles are obtained across different pathways, demonstrating the correctness of the technique, although significantly more sampling is required for the protein-ligand system. Our method offers a simple and effective way to incorporate quantum mechanical effects into computed free energies of binding

In Silico Study of Camptothecin-Based Pro-Drugs Binding to Human Carboxylesterase 2

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme–ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue

Molecular-dynamics simulations of liquid phase interfaces:understanding the structure of the glycerol/water-dodecane system

Modern spectroscopic techniques such as time-resolved second-harmonic-generation spectroscopy allow molecules to be examined selectively directly at phase interfaces. Two-phase systems formed by glycerol/water and alkane layers have previously been studied by time-resolved second-harmonic-generation spectroscopic measurements. In this molecular dynamics study, a triphenylmethane dye was inserted at the glycerol/water–alkane interface and was used as a probe for local properties such as viscosity. We now show how extensive simulations over a wide range of concentrations can be used to obtain a detailed view of the molecular structure at the glycerol/water–alkane interface. Glycerol is accumulated in a double layer adjacent to the alkane interface, which results in increased viscosity of the glycerol/water phase in the direct vicinity of the interface. We also show that conformational ensembles created by classical molecular-dynamics simulations can serve as input for QM/MM calculations, yielding further information such as transition dipoles, which can be compared with spectroscopic measurements