The distribution of tetramethylammonium ions on the surface of purple membranes

The two-dimensional distribution of deuterated tetramethylammonium (TMA⁺) ions on the surface of purple membranes of Halobacterium halobium was determined by neutron diffraction. The measurements were performed on stacks of these membranes with a high concentration of TMA⁺ molecules in the water layer between the membranes. A difference Fourier analysis of samples with deuterated and undeuterated ions showed an excess of 8.5 TMA⁺ ions per elementary cell in the lipid areas compared to the protein areas. A total number of 90 ions per elementary cell in the intermembrane space was estimated from the preparation procedure. The excess in the lipid domains may result from the higher affinity of TMA⁺ ions for the lipid head groups and/or from the fact that the protein (bacteriorhodopsin) protrudes slightly out of the lipid surface.Abbreviations BR bacteriorhodopsin - TMA Tetramethylammonium     

Capecitabine as a minor groove binder of DNA: molecular docking,molecular dynamics,and multi-spectroscopic studies

The interaction mechanism and binding mode of capecitabine with ctDNA was extensively investigated using docking and molecular dynamics simulations, fluorescence and circular dichroism (CD) spectroscopy, DNA thermal denaturation studies, and viscosity measurements. The possible binding mode and acting forces on the combination between capecitabine and DNA had been predicted through molecular simulation. Results indicated that capecitabine could relatively locate stably in the G-C base-pairs-rich DNA minor groove by hydrogen bond and several weaker nonbonding forces. Fluorescence spectroscopy and fluorescence lifetime measurements confirmed that the quenching was static caused by ground state complex formation. This phenomenon indicated the formation of a complex between capecitabine and ctDNA. Fluorescence data showed that the binding constants of the complex were approximately 2 × 10⁴ M^?1. Calculated thermodynamic parameters suggested that hydrogen bond was the main force during binding, which were consistent with theoretical results. Moreover, CD spectroscopy, DNA melting studies, and viscosity measurements corroborated a groove binding mode of capecitabine with ctDNA. This binding had no effect on B-DNA conformation.     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Drug‐binding energetics of human α‐1‐acid glycoprotein assessed by isothermal titration calorimetry and molecular docking simulations

This study utilizes sensitive, modern isothermal titration calorimetric methods to characterize the microscopic thermodynamic parameters that drive the binding of basic drugs to α‐1‐acid glycoprotein (AGP) and thereby rationalize the thermodynamic data in relation to docking models and crystallographic structures of the drug–AGP complexes. The binding of basic compounds from the tricyclic antidepressant series, together with miaserine, chlorpromazine, disopyramide and cimetidine, all displayed an exothermically driven binding interaction with AGP. The impact of protonation/deprotonation events, ionic strength, temperature and the individual selectivity of the A and F1*S AGP variants on drug‐binding thermodynamics was characterized. A correlation plot of the thermodynamic parameters for all of the test compounds revealed that an enthalpy–entropy compensation is in effect. The exothermic binding energetics of the test compounds were driven by a combination of favorable (negative) enthalpic (?Hº) and favorable (positive) entropic (?Sº) contributions to the Gibbs free energy (?Gº). Collectively, the data imply that the free energies that drive drug binding to AGP and its relationship to drug serum residency evolve from the complex interplay of enthalpic and entropic forces from interactions with explicit combinations of hydrophobic and polar side‐chain sub‐domains within the multi‐lobed AGP ligand binding cavity.Copyright © 2012 John Wiley & Sons, Ltd.     

Exploring the intercalation binding of tiamulin with DNA using multispectroscopy and a molecular modelling approach

The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity (η) and melting temperature (T_m) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant (K_a) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10⁴ M⁻¹) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10³ M⁻¹) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol⁻¹ using van der Waals forces as well as hydrogen bonds.     

Ion Permeation and Block of P2X2 Purinoceptors: Single Channel Recordings

P2X₂ purinoceptors are cation-selective channels activated by ATP and its analogues. Using single channel measurements we studied the channel's selectivity for the alkali metal ions and organic monovalent cations NMDG⁺, Tris⁺, TMA⁺, and TEA⁺. The selectivity sequence for currents carried by alkali metal ions is: K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, which is Eisenman sequence IV. This is different from the mobility sequence of the ions in free solution suggesting there is weak interaction between the ions and the channel interior. The relative conductance for alkali ions increases linearly in relation to the Stokes radius. The organic ions NMDG⁺, Tris⁺, TMA⁺ and TEA⁺ were virtually impermeant. The divalent ions (Mn²⁺, Mg²⁺, Ca²⁺ and Ba²⁺) induced a fast block visible as a reduction in amplitude of the unitary currents. Using a single-site binding model, the divalent ions exhibited an equilibrium affinity sequence of Mn²⁺ > Mg²⁺ > Ca²⁺ > Ba²⁺. Received: 3 May 1999/Revised: 23 August 1999     

Twisting DNA by salt

The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-molecule magnetic tweezer experiments and extensive all-atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Na⁺ < K⁺ < Rb⁺ < Ba²⁺ < Li⁺ ≈ Cs⁺ < Sr²⁺ < Mg²⁺ < Ca²⁺. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs⁺ and Sr²⁺. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.     

Combined spectroscopies and molecular docking approach to characterizing the binding interaction of enalapril with bovine serum albumin

The binding interaction between bovine serum albumin (BSA) and enalapril (ENPL) at the imitated physiological conditions (pH = 7.4) was investigated using UV–vis absorption spectroscopy (UV–vis), fluorescence emission spectroscopy (FES), synchronous fluorescence spectroscopy (SFS), Fourier transform infrared spectroscopy (FT‐IR), circular dichroism (CD) and molecular docking methods. It can be deduced from the experimental results from the steady‐state fluorescence spectroscopic titration that the intrinsic BSA fluorescence quenching mechanism induced by ENPL is static quenching, based on the decrease in the BSA quenching constants in the presence of ENPL with increase in temperature and BSA quenching rates >10¹⁰ L mol^?1 sec^?1. This result indicates that the ENPL–BSA complex is formed through an intermolecular interaction of ENPL with BSA. The main bonding forces for interaction of BSA and ENPL are van der Waal's forces and hydrogen bonding interaction based on negative values of Gibbs free energy change (ΔG ⁰), enthalpic change (ΔH ⁰) and entropic change (ΔS ⁰). The binding of ENPL with BSA is an enthalpy‐driven process due to |ΔH °| > |T ΔS °| in the binding process. The results of competitive binding experiments and molecular docking confirm that ENPL binds in BSA sub‐domain IIA (site I) and results in a slight change in BSA conformation, but BSA still retains its α‐helical secondary structure.     

Structure,dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK

The enzyme m¹A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNA^Leu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.     

Assessment on the binding characteristics of dasatinib,a tyrosine kinase inhibitor to calf thymus DNA: insights from multi-spectroscopic methodologies and molecular docking as well as DFT calculation

Abstract

The binding characteristics of calf thymus DNA (ct-DNA) with dasatinib (DSTN), a tyrosine kinase inhibitor was assessed through multi-spectroscopic methodologies and viscosity measurement combined with molecular docking as well as DFT calculation to understand the binding mechanism, affinity of DSTN onto ct-DNA, effect of DSTN on ct-DNA conformation, and among others. The results confirmed DSTN bound onto ct-DNA, leading to forming the DSTN–ct-DNA complex with the binding constant of 4.82?×?10³ M^?1 at 310?K. DSTN preferentially inserted to the minor groove of ct-DNA with rich A-T region, that was the binding mode of DSTN onto ct-DNA was groove binding. The enthalpic change (ΔH⁰) and entropic change (ΔS⁰) during the binding process of DSTN with ct-DNA were 128.9?kJ mol^?1 and 489.2?J mol^?1 K^?1, respectively, confirming clearly that the association of DSTN with ct-DNA was an endothermic process and the dominative driven-force was hydrophobic interaction. Meanwhile, the results also indicated that there was a certain extent of electrostatic force and hydrogen bonding, but they maybe play an auxiliary role. The CD measurement results confirmed the alteration in the helical configuration of ct-DNA but almost no change in the base stacking after binding DSTN. The results revealed that there was the obvious change in the conformation, the dipole moment, and the atomic charge distribution of DSTN in the B-DNA complexes, compared with free DSTN, to satisfy the conformational adaptation. From the obtained fronitier molecular orbitals of DSTN, it can be inferred that the nature of DSTN alters with the change of the environment around DSTN.

Communicated by Ramaswamy H. Sarma     

Congeneric but still distinct: how closely related trypsin ligands exhibit different thermodynamic and structural properties

A congeneric series of benzamidine-type ligands with a central proline moiety and a terminal cycloalkyl group—linked by a secondary amine, ether, or methylene bridge—was synthesized as trypsin inhibitors. This series of inhibitors was investigated by isothermal titration calorimetry, crystal structure analysis in two crystal forms, and molecular dynamics simulations. Even though all of these congeneric ligands exhibited essentially the same affinity for trypsin, their binding profiles at the structural, dynamic, and thermodynamic levels are very distinct. The ligands display a pronounced enthalpy/entropy compensation that results in a nearly unchanged free energy of binding, even though individual enthalpy and entropy terms change significantly across the series. Crystal structures revealed that the secondary amine-linked analogs scatter over two distinct conformational families of binding modes that occupy either the inside or of the outside the protein's S3/S4 specificity pocket. In contrast, the ether-linked and methylene-linked ligands preferentially occupy the hydrophobic specificity pocket. This also explains why the latter ligands could only be crystallized in the conformationally restricting closed crystal form whereas the derivative with the highest residual mobility in the series escaped our attempts to crystallize it in the closed form; instead, a well-resolved structure could only be achieved in the open form with the ligand in disordered orientation. These distinct binding modes are supported by molecular dynamics simulations and correlate with the shifting enthalpic/entropic signatures of ligand binding. The examples demonstrate that, at the molecular level, binding modes and thermodynamic binding signatures can be very different even for closely related ligands. However, deviating binding profiles provide the opportunity to optimally address a given target.     

Computational modeling and molecular dynamics simulations of mammalian cytoplasmic tyrosyl-tRNA synthetase and its complexes with substrates

Cytoplasmic tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. TyrRSs of pathogenic organisms have gained attention as potential targets for drug development. Identifying structural differences between various TyrRSs will facilitate the development of specific inhibitors for the TyrRSs of pathogenic organisms. However, there is a deficiency in structural data for mammalian cytoplasmic TyrRS in complexes with substrates. In this work, we constructed spatial structure of full-length Bos taurus TyrRS (BtTyrRS) and its complexes with substrates using the set of computational modeling techniques. Special attention was paid to BtTyrRS complexes with substrates [L-tyrosine, K⁺ and ATP:Mg²⁺] and intermediate products [tyrosyl-adenylate (Tyr-AMP), K⁺ and PP_i:Mg²⁺] with the different catalytic loop conformations. In order to analyze their dynamical properties, we performed 100 ns of molecular dynamics (MD) simulations. MD simulations revealed new structural data concerning the tyrosine activation reaction in mammalian TyrRS. Formation of strong interaction between Lys154 and γ-phosphate suggests the additional role of CP1 insertion as an important factor for ATP binding. The presence of a potassium-binding pocket within the active site of mammalian TyrRS compensates the absence of the second lysine in the KMSKS motif. Our data provide new details concerning a role of K⁺ ions at different stages of the first step of the tyrosylation reaction, including the coordination of substrates and involvement in the PP_i releasing. The results of this work suggest that differences between ATP-binding sites of mammalian and bacterial TyrRSs are meaningful and could be exploited in the drug design.     

Effects of the binding of calcium ions on the structure and dynamics of the ΦX174 virus investigated using molecular dynamics

It is known that the presence of calcium ions (Ca^2 +) is necessary for the enterobacterial virus ΦX174 to inject its DNA into the host cell, and that some mutations in the major capsid proteins lead to better survivability at higher temperatures. Our goal in the current study is to determine the physical changes in both the wild-type and mutant virus due to the binding of Ca^2 +. Thus, we performed molecular dynamics simulations of the ΦX174 major capsid protein complex with and without Ca^2 + bound. Our results show that binding of Ca^2 + leads to energetic and dynamical changes in the virus proteins. In particular, the results suggest that binding of Ca^2 + is energetically favorable and that the mutation leads to increased fluctuations of the protein complex (especially with the calcium ions bound to the complex), which may increase the rate of genome packaging and ejection for ΦX174.     

The thermodynamics of protein interactions with essential first row transition metals

BackgroundThe binding of metal ions to proteins is a crucial process required for their catalytic activity, structural stability and/or functional regulation. Isothermal titration calorimetry provides a wealth of fundamental information which when combined with structural data allow for a much deeper understanding of the underlying molecular mechanism.Scope of reviewA rigorous understanding of any molecular interaction requires in part an in-depth quantification of its thermodynamic properties. Here, we provide an overview of recent studies that have used ITC to quantify the interaction of essential first row transition metals with relevant proteins and highlight major findings from these thermodynamic studies.General significanceThe thermodynamic characterization of metal ion–protein interactions is one important step to understanding the role that metal ions play in living systems. Such characterization has important implications not only to elucidating proteins' structure-function relationships and biological properties but also in the biotechnology sector, medicine and drug design particularly since a number of metal ions are involved in several neurodegenerative diseases.Major conclusionsIsothermal titration calorimetry measurements can provide complete thermodynamic profiles of any molecular interaction through the simultaneous determination of the reaction binding stoichiometry, binding affinity as well as the enthalpic and entropic contributions to the free energy change thus enabling a more in-depth understanding of the nature of these interactions. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Binding Thermodynamics of Ferredoxin:NADP Reductase: Two Different Protein Substrates and One Energetics

The thermodynamics of the formation of binary and ternary complexes between Anabaena PCC 7119 FNR and its substrates, NADP⁺ and Fd, or Fld, has been studied by ITC. Despite structural dissimilarities, the main difference between Fd and Fld binding to FNR relates to hydrophobicity, reflected in different binding heat capacity and number of water molecules released from the interface. At pH 8, the formation of the binary complexes is both enthalpically and entropically driven, accompanied by the protonation of at least one ionizable group. His²⁹⁹ FNR has been identified as the main responsible for the proton exchange observed. However, at pH 10, where no protonation occurs and intrinsic binding parameters can be obtained, the formation of the binary complexes is entropically driven, with negligible enthalpic contribution. Absence of the FMN cofactor in Fld does not alter significantly the strength of the interaction, but considerably modifies the enthalpic and entropic contributions, suggesting a different binding mode. Ternary complexes show negative cooperativity (6-fold and 11-fold reduction in binding affinity, respectively), and an increase in the enthalpic contribution (more favorable) and a decrease in the entropic contribution (less favorable), with regard to the binary complexes energetics.     

Insights into molecular interactions between CaM and its inhibitors from molecular dynamics simulations and experimental data

In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca²⁺-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the “open” and “closed” conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM’s inhibitors correlated well with available experimental data as the r² obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca²⁺-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca²⁺-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca²⁺-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.     

Dynamics, Energetics, and Selectivity of the Low-K KcsA Channel Structure

Potassium channels are a diverse family of integral membrane proteins through which K⁺ can pass selectively. There is ongoing debate about the nature of conformational changes associated with the opening/closing and conductive/nonconductive states of potassium channels. The channels partly exert their function by varying their conductance through a mechanism known as C-type inactivation. Shortly after the activation of K⁺ channels, their selectivity filter stops conducting ions at a rate that depends on various stimuli. The molecular mechanism of C-type inactivation has not been fully understood yet. However, the X-ray structure of the KcsA channel obtained in the presence of low K⁺ concentration is thought to be representative of a K⁺ channel in the C-type inactivated state. Here, extensive, fully atomistic molecular dynamics and free-energy simulations of the low-K⁺ KcsA structure in an explicit lipid bilayer are performed to evaluate the stability of this structure and the selectivity of its binding sites. We find that the low-K⁺ KcsA structure is stable on the timescale of the molecular dynamics simulations performed, and that ions preferably remain in S1 and S4. In the absence of ions, the selectivity filter evolves toward an asymmetric architecture, as already observed in other computations of the high-K⁺ structure of KcsA and KirBac. The low-K⁺ KcsA structure is not permeable by Na⁺, K⁺, or Rb⁺, and the selectivity of its binding sites is different from that of the high-K⁺ structure.     

Enthalpy and entropy changes for the intercalation of small molecules to DNA. II. Ethidium and propidium fluoride

Enthalpy changes (ΔH_B) for the binding of ethidium (a monocation) and propidium (a dication) to calf thymus DNA have been determined calorimetrically in piperazine-N, N′-bis(2-ethanesulfonic acid) buffer with the fluoride ion as the counterion. Heats of dilution for the fluoride salts of ethidium and propidium were substantially less than the corresponding values found for other halide salts of these cations. At a Na⁺ ion concentrations of 0.019, ΔH_B = ?8.3 and ?7.9 ± 0.3 kcal mol^?1 for ethidium and propidium, respectively. For these two cations, just as was observed for the naphthalene monoimide (monocation) and diimide (dication) [H. P. Hopkins, K. A. Stevenson, and W. D. Wilson, (1986) J. Sol. Chem. 15 , 563–579], ΔH_B is within the same experimental error for both cations. Apparently, charge–charge interactions in DNA–cation complexes produce only small changes in the enthalpy for the system. In the concentration range 0.019–0.207, the ΔH_B values for propidium did not depend appreciably on the Na⁺ ion concentration, and a similar pattern was shown to exist for ethidium. When these results were combined with ΔG_B values for the binding of these cations to DNA, we found the variation of ΔS_B with Na⁺ ion concentration to be remarkably close to the predictions of modern polyelectrolyte theory, i.e., propidium binding to DNA causes approximately twice as many Na⁺ ions to be released into the bulk solution as does the binding of ethidium. The much stronger binding of propidium, relative to ethidium, at low ionic strengths is thus seen to be primarily due to entropic effects.