Showing papers on "Cooperative binding published in 2013"

Cooperative Binding

Abstract: Molecular binding is an interaction between molecules that results in a stable association between those molecules. Cooperative binding occurs if the number of binding sites of a macromolecule that are occupied by a specific type of ligand is a nonlinear function of this ligand's concentration. This can be due, for instance, to an affinity for the ligand that depends on the amount of ligand bound. Cooperativity can be positive (supralinear) or negative (infralinear). Cooperative binding is most often observed in proteins, but nucleic acids can also exhibit cooperative binding, for instance of transcription factors. Cooperative binding has been shown to be the mechanism underlying a large range of biochemical and physiological processes.

Allosteric mechanisms can be distinguished using structural mass spectrometry

Abstract: The activity of many proteins, including metabolic enzymes, molecular machines, and ion channels, is often regulated by conformational changes that are induced or stabilized by ligand binding. In cases of multimeric proteins, such allosteric regulation has often been described by the concerted Monod–Wyman–Changeux and sequential Koshland–Nemethy–Filmer classic models of cooperativity. Despite the important functional implications of the mechanism of cooperativity, it has been impossible in many cases to distinguish between these various allosteric models using ensemble measurements of ligand binding in bulk protein solutions. Here, we demonstrate that structural MS offers a way to break this impasse by providing the full distribution of ligand-bound states of a protein complex. Given this distribution, it is possible to determine all the binding constants of a ligand to a highly multimeric cooperative system, and thereby infer its allosteric mechanism. Our approach to the dissection of allosteric mechanisms relies on advances in MS—which provide the required resolution of ligand-bound states—and in data analysis. We validated our approach using the well-characterized Escherichia coli chaperone GroEL, a double-heptameric ring containing 14 ATP binding sites, which has become a paradigm for molecular machines. The values of the 14 binding constants of ATP to GroEL were determined, and the ATP-loading pathway of the chaperone was characterized. The methodology and analyses presented here are directly applicable to numerous other cooperative systems and are therefore expected to promote further research on allosteric systems.

Discovery of allosteric modulators of factor XIa by targeting hydrophobic domains adjacent to its heparin-binding site.

Abstract: To discover promising sulfated allosteric modulators (SAMs) of glycosaminoglycan-binding proteins (GBPs), such as human factor XIa (FXIa), we screened a library of 26 synthetic, sulfated quinazolin-4(3H)-ones (QAOs) resulting in the identification of six molecules that reduced the Vmax of substrate hydrolysis without influencing the KM. Mutagenesis of residues of the heparin-binding site (HBS) of FXIa introduced a nearly 5-fold loss in inhibition potency supporting recognition of an allosteric site. Fluorescence studies showed a sigmoidal binding profile indicating highly cooperative binding. Competition with a positively charged, heparin-binding polymer did not fully nullify inhibition suggesting importance of hydrophobic forces to binding. This discovery suggests the operation of a dual-element recognition process, which relies on an initial Coulombic attraction of anionic SAMs to the cationic HBS of FXIa that forms a locked complex through tight interaction with an adjacent hydrophobic patch. The dual-...

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity.

Abstract: The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of (15)N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 10(3) M(-1) and K2 = 3.4 ± 0.8 × 10(3) M(-1). Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.

Functional identification and characterization of sodium binding sites in Na symporters

Abstract: Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family Although the identification of Na+ in binding sites is beyond the resolution of the structures, two Na+ binding sites (Na1 and Na2) have been proposed in LeuT Na2 is conserved in the LeuT family but Na1 is not A biophysical method has been used to measure sodium dissociation constants (Kd) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na+ binding sites in hSGLT1 The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2 For the canonical Na2 site formed by two –OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium Kd by sixfold This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na+ and the binding of sugar In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na+ binding, and that Na+ binding to Na2 promotes binding to Na1 and also sugar binding

Structural and thermodynamic basis of (+)-α-pinene binding to human cytochrome P450 2B6.

Abstract: Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pin...

Binding of the 9-O-N-aryl/arylalkyl Amino Carbonyl Methyl Substituted Berberine Analogs to tRNAphe

Abstract: Background Three new analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNAphe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry. Methodology/Principal Findings Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNAphe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNAphe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNAphe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process. Conclusions/Significance The results showed that 9-O-N-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNAphe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNAphe.

Independent versus cooperative binding in polyethylenimine-DNA and Poly(L-lysine)-DNA polyplexes.

Abstract: The mechanism of polyethylenimine-DNA and poly(L-lysine)-DNA complex formation at pH 5.2 and 7.4 was studied by a time-resolved spectroscopic method. The formation of a polyplex core was observed to be complete at approximately N/P = 2, at which point nearly all DNA phosphate groups were bound by polymer amine groups. The data were analyzed further both by an independent binding model and by a cooperative model for multivalent ligand binding to multisubunit substrate. At pH 5.2, the polyplex formation was cooperative at all N/P ratios, whereas for pH 7.4 at N/P < 0.6 the polyplex formation followed independent binding changing to cooperative binding at higher N/Ps.

Mechanism of the interaction of plant alkaloid vincristine with DNA and chromatin: spectroscopic study.

Abstract: Chromatin has been successfully used as a tool for the study of genome function in cancers. Vincristine as a vinca alkaloid anticancer drug exerts its action by binding to tubulins. In this study the effect of vincristine on DNA and chromatin was investigated employing various spectroscopy techniques as well as thermal denaturation, equilibrium dialysis and DNA-cellulose affinity. The results showed that the binding of vincristine to DNA and chromatin reduced absorbance at both 260 and 210 nm with different extent. Chromopheres of chromatin quenched with the drug and fluorescence emission intensity decreased in a dose-dependent manner. Chromatin exhibited higher emission intensity changes compared to DNA. Upon addition of vincristine, Tm of DNA and chromatin exhibited hypochromicity without any shift in Tm. The binding of the drug induced structural changes in both positive and negative extremes of circular dichroism spectra and exhibited a cooperative binding pattern as illustrated by a positive slope observed in low r values of the binding isotherm. Vincristine showed higher binding affinity to double stranded DNA compared to single stranded one. The results suggest that vincristine binds with higher affinity to chromatin compared to DNA. The interaction is through intercalation along with binding to phosphate sugar backbone and histone proteins play fundamental role in this process. The binding of the drug to chromatin opens a new insight into vincristine action in the cell nucleus.

Use of (113)Cd NMR to probe the native metal binding sites in metalloproteins: an overview.

Abstract: Our laboratories have actively published in this area for several years and the objective of this chapter is to present as comprehensive an overview as possible. Following a brief review of the basic principles associated with (113)Cd NMR methods, we will present the results from a thorough literature search for (113)Cd chemical shifts from metalloproteins. The updated (113)Cd chemical shift figure in this chapter will further illustrate the excellent correlation of the (113)Cd chemical shift with the nature of the coordinating ligands (N, O, S) and coordination number/geometry, reaffirming how this method can be used not only to identify the nature of the protein ligands in uncharacterized cases but also the dynamics at the metal binding site. Specific examples will be drawn from studies on alkaline phosphatase, Ca(2+) binding proteins, and metallothioneins.In the case of Escherichia coli alkaline phosphatase, a dimeric zinc metalloenzyme where a total of six metal ions (three per monomer) are involved directly or indirectly in providing the enzyme with maximal catalytic activity and structural stability, (113)Cd NMR, in conjunction with (13)C and (31)P NMR methods, were instrumental in separating out the function of each class of metal binding sites. Perhaps most importantly, these studies revealed the chemical basis for negative cooperativity that had been reported for this enzyme under metal deficient conditions. Also noteworthy was the fact that these NMR studies preceded the availability of the X-ray crystal structure.In the case of the calcium binding proteins, we will focus on two proteins: calbindin D(9k) and calmodulin. For calbindin D(9k) and its mutants, (113)Cd NMR has been useful both to follow actual changes in the metal binding sites and the cooperativity in the metal binding. Ligand binding to calmodulin has been studied extensively with (113)Cd NMR showing that the metal binding sites are not directly involved in the ligand binding. The (113)Cd chemical shifts are, however, exquisitely sensitive to minute changes in the metal ion environment.In the case of metallothionein, we will reflect upon how (113)Cd substitution and the establishment of specific Cd to Cys residue connectivity by proton-detected heteronuclear (1)H-(113)Cd multiple-quantum coherence methods (HMQC) was essential for the initial establishment of the 3D structure of metallothioneins, a protein family deficient in the regular secondary structural elements of α-helix and β-sheet and the first native protein identified with bound Cd. The (113)Cd NMR studies also enabled the characterization of the affinity of the individual sites for (113)Cd and, in competition experiments, for other divalent metal ions: Zn, Cu, and Hg.

Mechanism and Kinetics of Acetyl-Lysine Binding to Bromodomains.

Abstract: Bromodomains are four-helix bundle proteins that specifically recognize acetylation of lysine side chains on histones. The available X-ray structures of bromodomain/histone tail complexes show that the conserved Asn residue in the loop between helices B and C is involved in a hydrogen bond with the acetyl-lysine side chain. Here we analyze the spontaneous binding of acetyl-lysine to the bromodomain TAF1(2) by the first molecular dynamics simulations of histone mark binding to an epigenetic reader protein. Multiple events of reversible association sampled along the unbiased simulations allow us to determine the pathway and kinetics of binding. The simulations show that acetyl-lysine has two major binding modes in TAF1(2) one of which corresponds to the available crystal structures and is stabilized by a hydrogen bond to the conserved Asn side chain. The other major binding mode is more buried than in the crystal structures and is stabilized by two hydrogen bonds with conserved residues of the loop between helices Z and A. In the more buried binding conformation, three of the six structured water molecules at the bottom of the binding pocket are displaced by the acetyl-lysine side chain. The kinetic analysis shows that the two binding modes interconvert on a faster time scale with respect to the association/dissociation process. The atomic-level description of the binding pathway and binding modes is useful for the design of small molecule modulators of histone binding to bromodomains.

Molecular Determinants of Binding to the Plasmodium Subtilisin-like Protease 1

Abstract: PfSUB1, a subtilisin-like protease of the human malaria parasite Plasmodium falciparum, is known to play important roles during the life cycle of the parasite and has emerged as a promising antimalarial drug target. In order to provide a detailed understanding of the origin of binding determinants of PfSUB1 substrates, we performed molecular dynamics simulations in combination with MM-GBSA free energy calculations using a homology model of PfSUB1 in complex with different substrate peptides. Key interactions, as well as residues that potentially make a major contribution to the binding free energy, are identified at the prime and nonprime side of the scissile bond and comprise peptide residues P4 to P2'. This finding stresses the requirement for peptide substrates to interact with both prime and nonprime side residues of the PfSUB1 binding site. Analyzing the energetic contributions of individual amino acids within the peptide-PfSUB1 complexes indicated that van der Waals interactions and the nonpolar part of solvation energy dictate the binding strength of the peptides and that the most favorable interactions are formed by peptide residues P4 and P1. Hot spot residues identified in PfSUB1 are dispersed over the entire binding site, but clustered areas of hot spots also exist and suggest that either the S4-S2 or the S1-S2' binding site should be exploited in efforts to design small molecule inhibitors. The results are discussed with respect to which binding determinants are specific to PfSUB1 and, therefore, might allow binding selectivity to be obtained.

Density-dependent cooperative non-specific binding in solid-phase SELEX affinity selection

Abstract: The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.

Structural changes in cytochrome c oxidase induced by binding of sodium and calcium ions: an ATR-FTIR study.

Abstract: Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to investigate the binding of Na+ and Ca2+cations to bovine cytochrome c oxidase in its fully oxidized and partially reduced, cyanide-ligated (a2+a33+-CN) (mixed valence) forms. These ions induced distinctly different IR binding spectra, indicating that the induced structural changes are different. Despite this, their binding spectra were mutually exclusive, confirming their known competitive binding behavior. Dissociation constants for Na+ and Ca2+ with the oxidized enzyme were 1.2 mM and 11 μM, respectively and Na+ binding appeared to involve cooperative binding of two Na+. Ca2+ binding induced a large IR spectrum, with prominent amide I/II polypeptide changes, bandshifts assigned to carboxylate and an arginine, and a number of bandshifts of heme a. The Na+-induced binding spectrum showed much weaker amide I/II and heme a changes but had similar shifts assignable to carboxylate and arginine residues. Yeast CcO also d...

An In Silico Analysis of the Binding Modes and Binding Affinities of Small Molecule Modulators of PDZ-Peptide Interactions

Abstract: Inhibitors of PDZ-peptide interactions have important implications in a variety of biological processes including treatment of cancer and Parkinson’s disease. Even though experimental studies have reported characterization of peptidomimetic inhibitors of PDZ-peptide interactions, the binding modes for most of them have not been characterized by structural studies. In this study we have attempted to understand the structural basis of the small molecule-PDZ interactions by in silico analysis of the binding modes and binding affinities of a set of 38 small molecules with known Ki or Kd values for PDZ2 and PDZ3 domains of PSD-95 protein. These two PDZ domains show differential selectivity for these compounds despite having a high degree of sequence similarity and almost identical peptide binding pockets. Optimum binding modes for these ligands for PDZ2 and PDZ3 domains were identified by using a novel combination of semi-flexible docking and explicit solvent molecular dynamics (MD) simulations. Analysis of the binding modes revealed most of the peptidomimectic ligands which had high Ki or Kd moved away from the peptide binding pocket, while ligands with high binding affinities remained in the peptide binding pocket. The differential specificities of the PDZ2 and PDZ3 domains primarily arise from differences in the conformation of the loop connecting βB and βC strands, because this loop interacts with the N-terminal chemical moieties of the ligands. We have also computed the MM/PBSA binding free energy values for these 38 compounds with both the PDZ domains from multiple 5 ns MD trajectories on each complex i.e. a total of 228 MD trajectories of 5 ns length each. Interestingly, computational binding free energies show good agreement with experimental binding free energies with a correlation coefficient of approximately 0.6. Thus our study demonstrates that combined use of docking and MD simulations can help in identification of potent inhibitors of PDZ-peptide complexes.

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites

Abstract: Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca2+ binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin–tropomyosin–actin complex. Herein, we report a calorimetric study of Ca2+ binding to HcTnC. Isotherms obtained at 25 °C (10 mM 2-morpholinoethanesulfonic acid, 50 mM KCl, pH 7.0) provided thermodynamic parameters for Ca2+ binding to both the high-affinity and the low-affinity domain of HcTnC. Ca2+ binding to the N-domain was shown to be endothermic in 2-morpholinoethanesulfonic acid buffer and allowed us to extract the thermodynamics of Ca2+ binding to the regulatory domain. This pattern stems from changes that occur at the Ca2+ site rather than structural changes of the protein. Molecular dynamics simulations performed on apo and calcium-bound HcTnC1–89 support this claim. The values of the Gibbs free energy for Ca2+ binding to the N-domain in the full-length protein and to the isolated domain (HcTnC1–89) are similar; however, differences in the entropic and enthalpic contributions to the free energy provide supporting evidence for the cooperativity of the C-domain and the N-domain. Thermograms obtained at two additional temperatures (10 and 37 °C) revealed interesting trends in the enthalpies and entropies of binding for both thermodynamic events. This allowed the determination of the change in heat capacity (∆Cp) from a plot of ∆H verses temperature and may provide evidence for positive cooperativity of Ca2+ binding to the C-domain.

Saccharide receptor achieves concentration dependent mannoside selectivity through two distinct cooperative binding pathways

Abstract: Tetrapodal receptor 1 relies upon structural flexibility to reveal new binding modes for saccharide recognition and to achieve unique pyranoside binding affinity and concentration dependent selectivity. The association constants, Kas, between 1 and eight pyranosides commonly found in cell surface glycans were measured in CDCl3 by 1H NMR titrations, revealing a preference for α- and β-octyl mannopyranosides (α-Man and β-Man). Whereas most of the pyranosides studied – α/β-octyl glucopyranoside (α/β-Glc), α/β-octyl galactopyranoside (α/β-Gal), and α/β-octyl N-acetylglucosaminopyranoside (α/β-GlcNAc) – bind 1 in a 1 : 1 stoichiometry at 25 °C, β-Man exclusively forms a 2 : 1 receptor–pyranoside complex. Alternatively, in an excess of pyranoside, 1 binds α- and β-Man in a 1 : 2 receptor : pyranoside stoichiometry with a high degree of positive cooperativity (K2/K1 ∼ 13.7 and 7.6 for α- and β-Man respectively) and selectivities as high as 16.8 : 1 α-Man : α-Gal. Moreover, this preference changes as a function of pyranoside concentration, favoring β-Glc at low concentration (<0.1 mM) and favoring mannosides at higher concentrations. The thermodynamic binding parameters (ΔH0 and ΔS0) reveal that the cooperativity in the second binding events drive the formation of 12:β-Man or 1:β-Man2 because of a decrease in unfavorable entropy upon each second binding event compared to the first. The structures of the complexes were determined by 1D and 2D 1H NMR spectroscopy in combination with molecular modeling. The 1:β-Man2 complex exhibits C2 symmetry, where both β-Man equivalents bind identical sites within 1, such that the pyranosides within the complex are symmetrically equivalent. Alternatively, 12:β-Man is a cage-like structure where only three of the aminopyrrolitic arms of the receptor are involved in binding, leaving a fourth available for further functionalization in later generation receptors. Multivalency and cooperativity are ubiquitous in Nature, and 1 utilizes these modes of recognition to achieve selectivity for monosaccharide residues.

A Novel Approach for the Evaluation of Positive Cooperative Guest Binding: Kinetic Consequences of Structural Tightening

Abstract: Cooperativity is one of the most relevant features displayed by biomolecules. Thus, one of the challenges in the field of supramolecular chemistry is to understand the mechanisms underlying cooperative binding effects. Traditionally, cooperativity has been related to multivalent receptors, but Williams et al. have proposed a different interpretation based on the strengthening of noncovalent interactions within receptors upon binding. According to such an interpretation, positive cooperative binding operates through structural tightening. Hence, a quite counterintuitive kinetic behavior for positively cooperative bound complexes may be postulated: the more stable the complex, the slower it is formed. Such a hypothesis was tested in a synthetic system in which positive cooperative binding was previously confirmed by calorimetric experiments. Indeed, a linear correlation between the thermodynamics (ΔG°) and the kinetics (ΔG(≠)) of guest binding confirmed the expected behavior. These distinctive kinetics provide solid evidence of positive cooperative guest binding, which is particularly useful bearing in mind that kinetic experiments are frequently and accurately carried out in both synthetic and biological systems.

Correlation between conformational equilibria of free host and guest binding affinity in non-preorganized receptors.

Abstract: Positive cooperativity between host conformational equilibria and guest binding has been widely reported in protein receptors. However, reported examples of this kind of cooperativity in synthetic hosts are scarce and largely serendipitous, among other things because it is hard to envision systems which display this kind of cooperativity. In order to shed some light on the correlation between conformational equilibria of free host and guest binding, selected structural modifications have been performed over a family of nonpreorganized hosts in order to induce conformational changes and to analyze their effect on the binding affinity. The conformational effect was evaluated by a theoretical conformational search and correlated with the ability of the receptors. All data suggest that those receptors that display the best association constants are able to sample folded conformations analogous to the conformational requirements for the binding of the guests. On the contrary, for those receptors where folded c...