Classification of water molecules in protein binding sites

Water molecules play a crucial role in mediating the interaction between a ligand and a macromolecular receptor. An understanding of the nature and role of each water molecule in the active site of a protein could greatly increase the efficiency of rational drug design approaches: if the propensity of a water molecule for displacement can be determined, then synthetic effort may be most profitably applied to the design of specific ligands with the displacement of this water molecule in mind. In this paper, a thermodynamic analysis of water molecules in the binding sites of six proteins, each complexed with a number of inhibitors, is presented. Two classes of water molecules were identified: those conserved and not displaced by any of the ligands, and those that are displaced by some ligands. The absolute binding free energies of 54 water molecules were calculated using the double decoupling method, with replica exchange thermodynamic integration in Monte Carlo simulations. It was found that conserved water molecules are on average more tightly bound than displaced water molecules. In addition, Bayesian statistics is used to calculate the probability that a particular water molecule may be displaced by an appropriately designed ligand, given the calculated binding free energy of the water molecule. This approach therefore allows the numerical assessment of whether or not a given water molecule should be targeted for displacement as part of a rational drug design strategy.     

Indolocarbazole-based foldamers capable of binding halides in water

A series of indolocarbazole-based foldamers have been prepared which can fold into a helical array with an internal cavity encircled by multiple indole NHs, thus allowing for binding anions by hydrogen bonds. The helical folding has been confirmed by computer modeling, 1H NMR spectroscopy, 2D ROESY experiments, and binding studies. A water-soluble derivative binds small, hydrophilic anions in the order Cl- > F- > Br- but negligibly with large, diffuse anions such as I- and ClO4-. Interestingly, the relative binding affinities of the fluoride and chloride ions in water are opposite to each other in an organic medium 4:1 (v/v) DMSO/MeOH, possibly due to the difference in the competing solvation energy.     

Hydration in drug design. 2. Influence of local site surface shape on water binding

Summary If water molecules are strongly bound at a protein-ligand interface, they are unlikely to be displaced during ligand binding. Such water molecules can change the shape of the ligand binding site and thus affect strategies for drug design. To understand the nature of water binding, and factors influencing it, water molecules at the ligand binding sites of 26 high-resolution protein-ligand complexes have been examined here. Water molecules bound in deep grooves and cavities between the protein and the ligand are located in the indentations on the protein-site surface, but not in the indentations on the ligand surface. The majority of the water molecules bound in deep indentations on the protein-site surface make multiple polar contacts with the protein surface. This may indicate a strong binding of water molecules in deep indentations on protein-site surfaces. The local shape of the site surface may influence the binding of water molecules that mediate protein-ligand interactions.     

A tight binding model for water

We demonstrate for the first time a tight binding model for water incorporating polarizable oxygen atoms. A novel aspect is that we adopt a "ground up" approach in that properties of the monomer and dimer only are fitted. Subsequently we make predictions of the structure and properties of hexamer clusters, ice-XI and liquid water. A particular feature, missing in current tight binding and semiempirical hamiltonians, is that we reproduce the almost two-fold increase in molecular dipole moment as clusters are built up toward the limit of bulk liquid. We concentrate on properties of liquid water, particularly dielectric constant and self diffusion coefficient, which are very well rendered in comparison with experiment. Finally we comment on the question of the contrasting densities of water and ice which is central to an understanding of the subtleties of the hydrogen bond.     

Tight binding and fluorescent sensing of oxalate in water

A dinuclear copper complex that binds tightly and selectively to oxalate over other dicarboxylates like malonate, succinate, and glutarate has been developed. This receptor can be used for fluorescent detection of oxalate in water at physicological pH by chemosensing ensemble approach. Crystal structure of oxalate bound to the receptor together with molecular mechanics and DFT computations provide insights into the tight and selective binding of the anion by the receptor.     

Degree of counterion binding on water in oil microemulsions

An ion-exchange process has been used to prepare HOT from NaOT (sodium bis(2-ethylhexyl)sulfosuccinate), where the Na(+) counterion has been replaced by H(+). The acidity function, H(0), of the aqueous core of HOT-based microemulsions decreases with W from H(0) approximately 0.6 at W>20 to H(0)=-1.4 at W=2. On the basis of the H(0) acidity function of the aqueous core and the dependence of H(0) on acid concentration, we have been able to determine the degree of counterion binding (beta) in microemulsions with a value of beta approximately 0.92 which is practically independent of the water content of the system.     

On the preparation of carbohydrate-protein conjugates using the traceless Staudinger ligation

[reaction: see text] The nature of a linker used for preparing glycoconjugate vaccines is of utmost importance as it may lead to immunogenic biomolecules. We report the conjugation of carbohydrate haptens to protein carriers leading to potential vaccines using the traceless Staudinger ligation. The ligation relies on the selective transfer of a phosphane substituent to an azide to form a native amide bond in the final product upon release of an oxidized phosphane byproduct. We designed new phosphino-functionalized cross-linkers suitable for protein carrier derivatization. We evaluated their utility in preparing conjugates using both synthetic and purified bacterial carbohydrates. The use of a borane-protected phosphane which is deprotected at the time of the ligation reaction led to the best results observed thus far in terms of stability toward oxidation and reactivity.     

Synthesis of amine-functionalized heparin oligosaccharides for the investigation of carbohydrate-protein interactions in microtiter plates

The synthesis of well-defined oligosaccharides is crucial for the establishment of structure-activity relationships for specific sequences of heparin, contributing to the understanding of the biological role of this polysaccharide. It is highly convenient that the synthetic oligosaccharides contain an orthogonal functional group that allows selective conjugation of the probes and expands their use as chemical tools in glycobiology. We present here the synthesis of a series of amine-functionalized heparin oligosaccharides using an n+2 modular approach. The conditions of the glycosylation reactions were carefully optimized to produce efficiently the desired synthetic intermediates with an N-benzyloxycarbonyl-protected aminoethyl spacer at the reducing end. The use of microwave heating greatly facilitates O- and N-sulfation steps, avoiding experimental problems associated with these reactions. The synthesized oligosaccharides were immobilized in 384-well microtiter plates and successfully probed with a heparin-binding protein, the basic fibroblast growth factor FGF-2. The use of hexadecyltrimethylammonium bromide minimized the amount of sugar required for attachment to the solid support. Using this approach we quantified heparin-protein interactions, and surface dissociation constants for the synthetic heparin derivatives were determined.     

Oxoanion binding by flexible guanidiniocarbonyl pyrrole-ammonium bis-cations in water

The syntheses of several bis-cations 1-5 with a simple primary ammonium cation attached via flexible linkers of varying length to a guanidiniocarbonyl pyrrole oxo anion binding site are reported. In UV-binding studies in aqueous buffer solution these bis-cations showed efficient binding of various N-acetyl amino acid carboxylates. However, complex affinity is significantly depending on both the anion and the length of the linker in the bis-cation. With increasing linker length, complex stability first increases until an optimum is reached for bis-cation 3 with a C4-linker. Then the complex stability decreases again. The best binding substrate in this series is N-acetyl phenyl alanine, most likely due to additional cation-pi-interactions between the aromatic ring and the guanidiniocarbonyl pyrrole cation. The formation of the complex between bis-cation 3 and N-acetyl phenyl alanine carboxylate was investigated further by fluorescence titrations and NOE studies, as well as molecular mechanics calculations.     

A designed receptor for pH-switchable ion binding in water

Molecules with conditional (switchable) properties are of considerable fundamental interest and are potentially useful for a broad range of applications, including chemical sensing. We have prepared a novel receptor, derived from the peracylation of cyclohexane 1,3,5-trimethanol with tyrosine, that suggests that the phenol-amine hydrogen bond may be an effective structural tool in the preparation of molecules with pH-switchable conformations. This conclusion is based on several key observations. First, the proton longitudinal relaxation rates for this molecule change in a pH-dependent fashion, while those for closely related structures do not. Second, NOESY spectra for the receptor change markedly depending on pH, with the spectrum acquired at pH 9.5 displaying NOEs consistent with the calculated "closed" conformation. Third, this molecule serves as a receptor for anions and cations in aqueous solution at high pH, but not at low pH, as demonstrated by UV-vis titrations and isothermal titration calorimetry (ITC).     

Free enthalpies of replacing water molecules in protein binding pockets

Water molecules in the binding pocket of a protein and their role in ligand binding have increasingly raised interest in recent years. Displacement of such water molecules by ligand atoms can be either favourable or unfavourable for ligand binding depending on the change in free enthalpy. In this study, we investigate the displacement of water molecules by an apolar probe in the binding pocket of two proteins, cyclin-dependent kinase 2 and tRNA-guanine transglycosylase, using the method of enveloping distribution sampling (EDS) to obtain free enthalpy differences. In both cases, a ligand core is placed inside the respective pocket and the remaining water molecules are converted to apolar probes, both individually and in pairs. The free enthalpy difference between a water molecule and a CH₃ group at the same location in the pocket in comparison to their presence in bulk solution calculated from EDS molecular dynamics simulations corresponds to the binding free enthalpy of CH₃ at this location. From the free enthalpy difference and the enthalpy difference, the entropic contribution of the displacement can be obtained too. The overlay of the resulting occupancy volumes of the water molecules with crystal structures of analogous ligands shows qualitative correlation between experimentally measured inhibition constants and the calculated free enthalpy differences. Thus, such an EDS analysis of the water molecules in the binding pocket may give valuable insight for potency optimization in drug design.     

Sequence-dependent binding of dipeptides by an artificial receptor in water

An artificial dipeptide receptor (1) was designed and observed to bind the deprotonated dipeptide Ac-D-Ala-D-Ala-OH in buffered water with K = 33,100 M(-1), whereas other dipeptides such as Ac-Gly-Gly-OH or Ac-D-Val-D-Val-OH were bound less efficiently, by factors of more than 10 (K < 3000 M(-1)). The efficient binding and the pronounced sequence selectivity are the result of a combination of strong electrostatic contacts and size-discriminating hydrophobic interactions. To provide such a combination, a guanidiniocarbonylpyrrole cation was attached to a novel cyclotribenzylene-substituted alanine derivative 5, to provide a hydrophobic bowl-shaped cavity just large enough to bind a methyl group but not any larger alkyl chains, thus causing the receptor to prefer alanine to valine. We describe the synthesis of 1 and the evaluation of its complexation properties in UV and fluorescence titration studies.     

Negatively charged crown ethers for binding paraquat in water

A water soluble negatively charged fluorescent 1,4-benzo-1,5-naphtho-36-crown-10-based host has been devised and synthesized.As shown by proton NMR,ESI mass spectrometry and UV-vis spectroscopy,it binds paraquat with a 1:1 stoichiometry and an association constant of 4.50(±0.02) ×103 M-1 in water.Its complexation with paraquat in water was further investigated by fluorescence emission spectroscopy.The results revealed that when paraquat was added to the water solution of the host,the fluorescence emission o...     

Small cavitands specifically binding a water molecule

[structure: see text] Three new C(2v) cavitands based on resorcin[4]arene bind water specifically at low temperature in CD(2)Cl(2) or CDCl(3) due to their complementarity to water as well as the solvophobic interaction. The averaged DeltaH(o) and DeltaS(o) values are -2.3 kcal mol(-1) and -128 cal mol(-1) K(-1), which gave the averaged -DeltaG(o) of 1.9 kcal mol(-1) at -50 degrees C in water saturated CD(2)Cl(2).     

Facile fabrication of branched-chain carbohydrate chips for studying carbohydrate-protein interactions by QCM biosensor

A novel approach for fabricating branched-chain (BC) carbohydrate chips to study carbohydrate-protein interactions using quantz crystal microbalance (QCM) biosensor was developed. This approach utilizes functional alkynyl-branch molecule modified chip surfaces, which is functionalized with terminal alkynyl group for covalent linking of unprotected azide-carbohydrates via click chemistry.     

Ligand design by targeting a binding site water

Solvent reorganization is a major driving force of protein–ligand association, but the contribution of binding site waters to ligand affinity is poorly understood. We investigated how altered interactions with a water network can influence ligand binding to a receptor. A series of ligands of the A_2A adenosine receptor, which either interacted with or displaced an ordered binding site water, were studied experimentally and by molecular dynamics simulations. An analog of the endogenous ligand that was unable to hydrogen bond to the ordered water lost affinity and this activity cliff was captured by molecular dynamics simulations. Two compounds designed to displace the ordered water from the binding site were then synthesized and evaluated experimentally, leading to the discovery of an A_2A agonist with nanomolar activity. Calculation of the thermodynamic profiles resulting from introducing substituents that interacted with or displaced the ordered water showed that the gain of binding affinity was enthalpy driven. Detailed analysis of the energetics and binding site hydration networks revealed that the enthalpy change was governed by contributions that are commonly neglected in structure-based drug optimization. In particular, simulations suggested that displacement of water from a binding site to the bulk solvent can lead to large energy contributions. Our findings provide insights into the molecular driving forces of protein–ligand binding and strategies for rational drug design.

Solvent reorganization is a major driving force of protein–ligand association, but the contribution of binding site waters to ligand affinity is poorly understood.     

Self consistent tight binding model for dissociable water

We report results of development of a self consistent tight binding model for water. The model explicitly describes the electrons of the liquid self consistently, allows dissociation of the water and permits fast direct dynamics molecular dynamics calculations of the fluid properties. It is parameterized by fitting to first principles calculations on water monomers, dimers, and trimers. We report calculated radial distribution functions of the bulk liquid, a phase diagram and structure of solvated protons within the model as well as ac conductivity of a system of 96 water molecules of which one is dissociated. Structural properties and the phase diagram are in good agreement with experiment and first principles calculations. The estimated DC conductivity of a computational sample containing a dissociated water molecule was an order of magnitude larger than that reported from experiment though the calculated ratio of proton to hydroxyl contributions to the conductivity is very close to the experimental value. The conductivity results suggest a Grotthuss-like mechanism for the proton component of the conductivity.