Network Of Side Chains Research Articles

On the Structural Basis of Modal Gating Behavior in K + Channels

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K+ channel KcsA displays three distinct gating modes, high-Po, low-Po and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. In KcsA, the hydrogen bond network between Glu71, Asp80 and Trp67 that surrounds the selectivity filter has been shown to regulate C-type inactivation, with the Glu71-Asp80 pair having the strongest influence on selectivity filter stability. Here, we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side-chain-specific way which mirror those seen in wild-type channels. Results from high-resolution crystal structures along with molecular dynamic simulations suggest that specific interactions in the side-chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. These findings highlight the key role of the selectivity filter in regulating modal gating behavior in K+ channels.

On the structural basis of modal gating behavior in K+ channels

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K+ channel KcsA displays three distinct gating modes, high-Po, low-Po and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here, we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side-chain-specific way. These gating modes mirror those seen in wild–type channels and suggest that specific interactions in the side-chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K+ channels.

Specificity for Homooligomer versus Heterooligomer Formation in Integrin Transmembrane Helices

Transmembrane (TM) helices engage in homomeric and heteromeric interactions that play essential roles in the folding and assembly of TM proteins. However, features that explain their propensity to interact homomerically or heteromerically and determine the strength of these interactions are poorly understood. Integrins provide an ideal model system for addressing these questions because the TM helices of full-length integrins interact heteromerically when integrins are inactive, but isolated TM helices are also able to form homodimers or homooligomers in micelles and bacterial membranes. We sought to determine the features defining specificity for homointeractions versus heterointeractions by conducting a comprehensive comparison of the homomeric and heteromeric interactions of integrin αIIbβ3 TM helices in biological membranes. Using the TOXCAT assay, we found that residues V700, M701, A703, I704, L705, G708, L709, L712, and L713, which are located on the same face of the β3 helix, mediate homodimer formation. We then characterized the β3 heterodimer by measuring the ability of β3 helix mutations to cause ligand binding to αIIbβ3. We found that mutating V696, L697, V700, M701, A703. I704, L705, G708, L712, and L713, but not the small residue–X3–small residue motif S699–X3–A703, caused constitutive αIIbβ3 activation, as well as persistent focal adhesion kinase phosphorylation dependent on αIIbβ3 activation. Because αIIb and β3 use the same face of their respective TM helices for homomeric and heteromeric interactions, the interacting surface on each has an intrinsic “stickiness” predisposing towards helix–helix interactions in membranes. The residues responsible for heterodimer formation comprise a network of interdigitated side chains with considerable geometric complementarity; mutations along this interface invariably destabilize heterodimer formation. By contrast, residues responsible for homomeric interactions are dispersed over a wider surface. While most mutations of these residues are destabilizing, some stabilized homooligomer formation. We conclude that the αIIbβ3 TM heterodimer shows the hallmark of finely tuned heterodimeric interaction, while homomeric interaction is less specific.

Structure-Function Analysis of Inositol Hexakisphosphate-induced Autoprocessing in Clostridium difficile Toxin A

The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.

Structural Insight into PPARγ Activation Through Covalent Modification with Endogenous Fatty Acids

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor that regulates lipid homeostasis, and several fatty acid metabolites have been identified as PPARγ ligands. Here, we present four crystal structures of the PPARγ ligand binding domain (LBD) covalently bound to endogenous fatty acids via a unique cysteine, which is reportedly critical for receptor activation. The structure analyses of the LBD complexed with 15-deoxy-Δ 12,14-prostaglandin J 2 (15d-PGJ 2) revealed that the covalent binding of 15d-PGJ 2 induced conformational changes in the loop region following helix H2′, and rearrangements of the side-chain network around the created covalent bond in the LBD. Point mutations of these repositioned residues on the loop and helix H3 almost completely abolished PPARγ activation by 15d-PGJ 2, indicating that the observed structural alteration may be crucial for PPARγ activation by the endogenous fatty acid. To address the issue of partial agonism of endogenous PPARγ ligands, we took advantage of a series of oxidized eicosatetraenoic acids (oxoETEs) as covalently bound ligands to PPARγ. Despite similar structural and chemical properties, these fatty acids exhibited distinct degrees of transcriptional activity. Crystallographic studies, using two of the oxoETE/PPARγ LBD complexes, revealed that transcriptional strength of each oxoETE is associated with the difference in the loop conformation, rather than the interaction between each ligand and helix H12. These results suggest that the loop conformation may be responsible for the modulation of PPARγ activity. Based on these results, we identified novel agonists covalently bound to PPARγ by in silico screening and a cell-based assay. Our crystallographic study of LBD complexed with nitro-233 demonstrated that the expected covalent bond is indeed formed between this newly identified agonist and the cysteine. This study presents the structural basis for the activation and modulation mechanism of PPARγ through covalent modification with endogenous fatty acids.

Congenital disease SNPs target lineage specific structural elements in protein kinases

The catalytic domain of protein kinases harbors a large number of disease-causing single nucleotide polymorphisms (SNPs) and common or neutral SNPs that are not known or hypothesized to be associated with any disease. Distinguishing these two types of polymorphisms is critical in accurately predicting the causative role of SNPs in both candidate gene and genome-wide association studies. In this study, we have analyzed the structural location of common and disease-associated SNPs in the catalytic domain of protein kinases and find that, although common SNPs are randomly distributed within the catalytic core, known disease SNPs consistently map to regulatory and substrate binding regions. In particular, a buried side-chain network that anchors the flexible activation loop to the catalytic core is frequently mutated in disease patients. This network was recently shown to be absent in distantly related eukaryotic-like kinases, which lack an exaggerated activation loop and, presumably, are not regulated by phosphorylation.

De Novo Computational Design of Retro-Aldol Enzymes

The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.

Mechanism of Proton Transfer in the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

Abstract 3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of DV and D2OV for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, 2H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys159 changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on Vmax and V/Kandrosterone and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on Vmax is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Bronsted relationship between the log(V/Kd-baseEt) of the external amine (corrected for molecular size effects) and pKa is linear for the K159A mutant-catalyzed reaction at pH 10.4 (β = 0.85 ± 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr155 to Lys159 via 2′-OH of ribose.

Effect of Nematic Order Coupling on the Phase Diagrams of Side-Chain Polymer Gels with Liquid Crystal Solvent

AbstractWe have explored the influence of nematic order coupling on the swelling and phase diagram of polymer networks in nematogenic low molar weight LC solvent, in view of the increasing importance of such systems in advanced optical devices. Firstly, one isotropic polyacrylate network and one nematic sidechain polyacrylate network is prepared. Immersing these two networks in nematic LC solvent lead to the formation of well-defined polymer gels with LC solvents and allow us to establish the phase diagram of these relatively new materials in the concentration-temperature frame. Results were critically analysed as a function of temperature and network nature (isotropic or nematic). We demonstrate that in the case of gel made from an isotropic polymer network, the LC solvents fails to form a nematic phase inside the gel due to the lack of anisotropic coupling. Moreover, in that case the gel shrinks below TNI Solvent because of a strong “entropic” incompatibility between the isotropic flexible coils and the LC nematogens. However, the situation is drastically different in the case of LC side-chain network due to a strong coupling of the nematic order between the mesogens of the solvents and those of the polymer backbone. In that case, tremendous distortions appear in the phase-diagram, in which we especially emphasize the apparition of a stable nematic gel phase and a miscibility gap between TNI Solvent and TNI Gel. Finally, results are critically examined and compared to the few studies found in the literature.

NMR Dynamic Studies Suggest that Allosteric Activation Regulates Ligand Binding in Chicken Liver Bile Acid-binding Protein

Apo chicken liver bile acid-binding protein has been structurally characterized by NMR. The dynamic behavior of the protein in its apo- and holo-forms, complexed with chenodeoxycholate, has been determined via (15)N relaxation and steady state heteronuclear (15)N((1)H) nuclear Overhauser effect measurements. The dynamic parameters were obtained at two pH values (5.6 and 7.0) for the apoprotein and at pH 7.0 for the holoprotein, using the model free approach. Relaxation studies, performed at three different magnetic fields, revealed a substantial conformational flexibility on the microsecond to millisecond time scales, mainly localized in the C-terminal face of the beta-barrel. The observed dynamics are primarily caused by the protonation/deprotonation of a buried histidine residue, His(98), located on this flexible face. A network of polar buried side chains, defining a spine going from the E to J strand, is likely to provide the long range connectivity needed to communicate motion from His(98) to the EF loop region. NMR data are accompanied by molecular dynamics simulations, suggesting that His(98) protonation equilibrium is the triggering event for the modulation of a functionally important motion, i.e. the opening/closing at the protein open end, whereas ligand binding stabilizes one of the preexisting conformations (the open form). The results presented here, complemented with an analysis of proteins belonging to the intracellular lipid-binding protein family, are consistent with a model of allosteric activation governing the binding mechanism. The functional role of this mechanism is thoroughly discussed within the framework of the mechanism for the enterohepatic circulation of bile acids.

Molecular basis of Toxoplasma gondii atovaquone resistance modeled in Saccharomyces cerevisiae

0 d ondii is a widespread disease affecting primarily immunocomromised and pregnant individuals [1]. Atovaquone is a recently ntroduced anti-malarial compound with broad spectrum activty against various apicomplexan parasites [2–5] including T. ondii [6]. Approved by the FDA in 1995, this drug is a potent nd specific inhibitor of the cytochrome bc1 complex [7], an ssential respiratory enzyme present in the inner mitochondrial embrane. Two recent studies on treatment of malaria during regnancy show that atovaquone is a remarkably well tolerated olecule, safe for both the mother and the fetus [8,9]. Unforunately, there is strong evidence that the targeted parasites are ble to spontaneously develop drug resistance by mutation of mino acid residues located in or near the atovaquone-binding ite on cytochrome b. Because the yeast bc1 complex is also trongly inhibited by atovaquone, we have previously develped Saccharomyces cerevisiae as a model to study cytochrome mutations conferring atovaquone resistance in Pneumocystis 10,11] and Plasmodium species [12]. In the present study, we ave successfully transferred two mutations associated with atoaquone resistance in T. gondii [13] into the yeast cytochrome to the one recently found in the crystal structure of the yeast bc1 complex with a hydroxy-benzoxythiazol bound at center P [14]. The hydroxyl group of the hydroxy-naphthoquinone binds via a hydrogen bond to the nitrogen of His-181 of the Rieske iron–sulfur protein. On the opposite side of the ring system the carbonyl group at position 4 of the quinone ring of atovaquone forms a water-mediated hydrogen bond with Glu-272 of cytochrome b. The bulk of the molecular interactions between atovaquone and cytochrome b are essentially hydrophobic with a network of aromatic and aliphatic side chains surrounding the inhibitor. There is a high score of cytochrome b sequence identity (about 70%) within the atovaquone binding pocket between S. cerevisiae and T. gondii (Fig. 1A). We have thus chosen the yeast to study how mutations can affect the drug efficacy. Both atovaquone-resistant mutations in the cytochrome b sequence previously identified in T. gondii were localized within this binding site [13]. These two mutations (M129L and I254L in T. gondii numbering, equivalent to M139L and I269L in the yeast numbering system) associated with resistance in T. gondii gene. This has allowed us to biochemically confirm the linkge of atovaquone resistance to the cytochrome b mutations and o predict at the molecular level the mechanism by which T. ondiimay counter the efficacy of this potential anti-toxoplasma rug. were transferred into the S. cerevisiae cytochrome b gene by the biolistic method [11]. Growth of the two mutated strains was monitored in a nonfermentable medium in order to study the impact of the mutations on the yeast cells’ respiration. The growth curves revealed t s n o v m The molecular target of atovaquone is now known to be he ubiquinol oxidation pocket at center P of the cytochrome

Invariant amino acids essential for decoding function of polypeptide release factor eRF1.

In eukaryotic ribosome, the N domain of polypeptide release factor eRF1 is involved in decoding stop signals in mRNAs. However, structure of the decoding site remains obscure. Here, we specifically altered the stop codon recognition pattern of human eRF1 by point mutagenesis of the invariant Glu55 and Tyr125 residues in the N domain. The 3D structure of generated eRF1 mutants was not destabilized as demonstrated by calorimetric measurements and calculated free energy perturbations. In mutants, the UAG response was most profoundly and selectively affected. Surprisingly, Glu55Arg mutant completely retained its release activity. Substitution of the aromatic ring in position 125 reduced response toward all stop codons. This result demonstrates the critical importance of Tyr125 for maintenance of the intact structure of the eRF1 decoding site. The results also suggest that Tyr125 is implicated in recognition of the 3d stop codon position and probably forms an H-bond with Glu55. The data point to a pivotal role played by the YxCxxxF motif (positions 125–131) in purine discrimination of the stop codons. We speculate that eRF1 decoding site is formed by a 3D network of amino acids side chains.

S641 contributes HERG K+ channel inactivation.

The kinetics of voltage-dependent inactivation of the rapidly activating delayed rectifier, IKr, are unique among K+ channels. The human ether-a-gogo-related gene (HERG) encodes the pore-forming subunit of IKr and shares a high degree of homology with ether-a-gogo (EAG) channels that do not inactivate. Within those segments thought to contribute to the channel pore, HERG possesses several serine residues that are not present in EAG channels. Two of these serines, S620 and S631, are known to be required for inactivation. We now show that a third serine, S641, which resides in the outer portion of the sixth transmembrane segment, is also critical for normal inactivation. As with the other serines, S641 is also involved in maintaining ion selectivity of the HERG channel and alters sensitivity to block by E4031. Larger charged or polar substitutions (S641D and S641T) disrupted C-type inactivation in HERG. Smaller aliphatic and more conservative substitutions (S641A and S641C) facilitated C-type inactivation. Our data show that, like S620 and S631, S641 is another key residue for the rapid inactivation. The altered inactivation of mutations at S620, S631, and S641 were dominant, suggesting that a network of hydroxyl side chains is required for the unique inactivation, permeation, and rectification of HERG channels.

New insights into intracellular lipid binding proteins: The role of buried water.

The crystal structures of most intracellular lipid binding proteins (LBPs) show between 5 and 20 internally bound water molecules, depending on the presence or the absence of ligand inside the protein cavity. The structural and functional significance of these waters has been discussed for several LBPs based on studies that used various biophysical techniques. The present work focuses on two very different LBPs, heart-type fatty acid binding protein (H-FABP) and ileal lipid binding protein (ILBP). Using high-resolution nuclear magnetic resonance spectroscopy, certain resonances belonging to side-chain protons that are located inside the water-filled lipid binding cavity were observed. In the case of H-FABP, the pH- and temperature-dependent behavior of selected side-chain resonances (Ser82 OgH and the imidazole ring protons of His93) indicated an unusually slow exchange with the solvent, implying that the intricate hydrogen-bonding network of amino-acid side-chains and water molecules in the protein interior is very rigid. In addition, holo H-FABP appeared to display a reversible self-aggregation at physiological pH. For ILBP, on the other hand, a more solvent-accessible protein cavity was deduced based on the pH titration behavior of its histidine residues. Comparison with data from other LBPs implies that the evolutionary specialization of LBPs for certain ligand types was not only because of mutations of residues directly involved in ligand binding but also to a refinement of the internal water scaffold.

Structure of Tagatose-1,6-bisphosphate Aldolase: INSIGHT INTO CHIRAL DISCRIMINATION, MECHANISM, AND SPECIFICITY OF CLASS II ALDOLASES

Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate. The high resolution (1.45 A) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined. Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer. A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations. An unusual Na+ binding site is created using a pi interaction with Tyr183 in addition to five oxygen ligands. The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens. Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism. Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases. The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme. The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).

Conformational Effects in Mesogenic Fragments in Liquid-Crystalline Side-Chain Polymers and Networks

Side-chain liquid-crystalline polymers (linear and cross-linked) containing alkoxycyanobiphenyl as a mesogenic fragment were studied in solution and in bulk by FTIR spectroscopy. It was found for linear polymers that the transition from solution to bulk is accompanied by the appearance of new bands (satellites) at both sides of the C⋮N mode in the FTIR spectrum. Introducing nonmesogenic fragments into copolymer together with the network formation does not affect their number and frequencies but changes their relative intensities. The appearance of satellites is assumed to be connected with the appearance of conformational inhomogeneity of the aromatic cores of mesogenic fragments by the interaction of their dipoles. This assumption was confirmed by quantum chemistry calculations of model molecules.

Sulfite reductase structure at 1.6 A: evolution and catalysis for reduction of inorganic anions.

Fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen are catalyzed by sulfite and nitrite reductases. The crystallographic structure of Escherichia coli sulfite reductase hemoprotein (SiRHP), which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia, was solved with multiwavelength anomalous diffraction (MAD) of the native siroheme and Fe4S4 cluster cofactors, multiple isomorphous replacement, and selenomethionine sequence markers. Twofold symmetry within the 64-kilodalton polypeptide generates a distinctive three-domain alpha/beta fold that controls cofactor assembly and reactivity. Homology regions conserved between the symmetry-related halves of SiRHP and among other sulfite and nitrite reductases revealed key residues for stability and function, and identified a sulfite or nitrite reductase repeat (SNiRR) common to a redox-enzyme superfamily. The saddle-shaped siroheme shares a cysteine thiolate ligand with the Fe4S4 cluster and ligates an unexpected phosphate anion. In the substrate complex, sulfite displaces phosphate and binds to siroheme iron through sulfur. An extensive hydrogen-bonding network of positive side chains, water molecules, and siroheme carboxylates activates S-O bonds for reductive cleavage.

New tubular single-stranded helix of poly-L-amino acids suggested by molecular mechanics calculations: I. Homopolypeptides in isolated environments

A search was made in terms of molecular mechanics calculation for tubular, or pore-forming, single-stranded helices of poly-L-amino acids. Such a helix was found in the vicinity of (phi, psi, omega) = 81 degrees, 98 degrees, 170 degrees) in the conformational space. It was the 6.2(20) helix of right-handedness. The 6.2(20) helix, here named the "mu helix," had a cylindrical pore along its helical axis. The diameter of the pore was 6.6 A on the basis of the atom centers of carbonyl carbons and amino nitrogens. The left-handed mu helix was less stable than the right-handed counterpart. The conformation energy of the mu helix, expressed relative to that of the alpha helix of the same polypeptide, depended to a great extent on amino acid composition. It varied over a range of a few kilocalories per mol per residue above and below the conformation energy of the alpha helix of the same polypeptide. This marked diversity in the relative conformation energy of the mu helix can be ascribed primarily to the difference in the relative position of alpha-carbons between the mu and the alpha helices. The conformational entropy made only a small contribution, if any, to the relative stability of the mu helix. There was a hydrogen-bonded network of side chains in the mu helices of poly-L-glutamine and poly-L-asparagine.

Three‐dimensional structure of a fluorescein–Fab complex crystallized in 2‐methyl‐2,4‐pentanediol

The crystal structure of a fluorescein-Fab (4-4-20) complex was determined at 2.7 A resolution by molecular replacement methods. The starting model was the refined 2.7 A structure of unliganded Fab from an autoantibody (BV04-01) with specificity for single-stranded DNA. In the 4-4-20 complex fluorescein fits tightly into a relatively deep slot formed by a network of tryptophan and tyrosine side chains. The planar xanthonyl ring of the hapten is accommodated at the bottom of the slot while the phenylcarboxyl group interfaces with solvent. Tyrosine 37 (light chain) and tryptophan 33 (heavy chain) flank the xanthonyl group and tryptophan 101 (light chain) provides the floor of the combining site. Tyrosine 103 (heavy chain) is situated near the phenyl ring of the hapten and tyrosine 102 (heavy chain) forms part of the boundary of the slot. Histidine 31 and arginine 39 of the light chain are located in positions adjacent to the two enolic groups at opposite ends of the xanthonyl ring, and thus account for neutralization of one of two negative charges in the haptenic dianion. Formation of an enol-arginine ion pair in a region of low dielectric constant may account for an incremental increase in affinity of 2-3 orders of magnitude in the 4-4-20 molecule relative to other members of an idiotypic family of monoclonal antifluorescyl antibodies. The phenyl carboxyl group of fluorescein appears to be hydrogen bonded to the phenolic hydroxyl group of tyrosine 37 of the light chain. A molecule of 2-methyl-2,4-pentanediol (MPD), trapped in the interface of the variable domains just below the fluorescein binding site, may be partly responsible for the decrease in affinity for the hapten in MPD.