Hydrophilic Reagents Research Articles

Newly Designed Modifier Prolongs the Action of Short-Lived Peptides and Proteins by Allowing Their Binding to Serum Albumin

We found that human serum albumin (HSA) contains a single binding domain for derivatives of long-chain fatty acid (LCFA)-like molecules in which the carboxylate is replaced by sulfonate. Accordingly, we have synthesized 16-sulfo-hexadecanoic acid-N-hydroxysuccinimide ester [HO(3)S-(CH(2))(15)-CONHS], an agent that reacts selectively with the amino side chains of peptides and proteins. A macromolecule containing a single 16-sulfohexadecanoate moiety associating with albumin with a K(a) value of 0.83 ± 0.08 × 10(6) M(-1), a sufficient affinity to extend the actions in vivo of such short-lived peptides and proteins. Subcutaneous administration of insulin-NHCO-(CH(2))(15)-SO(3)(-) into mice facilitated a glucose-lowering effect 4.3 times in duration and 6.6 times in area under the curve (AUC) as compared to an in vitro equipotent amount of Zn(2+)-free insulin. Similarly, subcutaneous and intravenous administration of exendin-4-NHCO-(CH(2))(15)-SO(3)(-) to mice yielded prolonged and stable reduction in glucose level, 5-9-fold longer than that of exendin-4. Also, a single subcutaneous administration of human interferon-α2-[NH-CO-(CH(2))(15)-SO(3)(-)](3) to mice yielded circulating antiviral activity over a period of 40 h. In conclusion, a simple, hydrophilic reagent has been engineered, synthesized, and studied. Its linkage to peptides and proteins in a monomodified fashion yielded hydrophilic, prolonged acting derivatives, due to their acquired ability to associate with serum albumin after administration.

Bringing trees into the fuel line

Bringing trees into the fuel line

Gating Ion Translocation through the Na,K-ATPase Pump

Na,K pumps behave like channels controlled by extracellular- and cytoplasmic-side gates driven to open and close alternately by coupling to a cycle of Na-mediated phosphorylation and K-mediated dephosphorylation. The marine agent palytoxin disrupts that coupling, allowing both gates to sometimes be open, thereby transforming pumps into channels. So, palytoxin enables ion-pathway characterization and assays of gating mechanisms with single-molecule resolution. Palytoxin-bound Na,K pump-channels exposed to Na solutions and millimolar cytoplasmic ATP spend ∼90% of the time open, each channel conducting millions of Na ions per second. Cysteine scanning of the transmembrane domain with small hydrophilic reagents reveals an unbranched cation-selective pathway crossing the entire membrane. Replacing external Na with K promptly shuts the extracellular-side gate, like during K transport by unmodified pumps. The high open probability of palytoxin-bound pump-channels in Na solutions falls ≥5 fold if ATP is withdrawn. Cytoplasmic AMPPNP or ADP can replace ATP - all acting with low affinity - in supporting this cytoplasmic-side gate opening, echoing acceleration of cytoplasmic K release by these nucleotides in unmodified Na,K pumps. This low-affinity, modulatory, nucleotide binding occurs electrostatically distant from the normally-phosphorylated Asp, as its mutation to Asn, Ala, or Glu leaves nucleotide affinity unaltered. The phosphate analogs BeFx or AlFx close palytoxin-bound pump-channels, apparently in all-or-none manner, quickly when ATP is absent, but slowly in millimolar ATP. This antagonism reflects modulatory nucleotide binding because ADP or AMPPNP mimic it, and it persists after mutating the phosphate-acceptor Asp to Asn. Thus BeFx or AlFx binds most tightly to pump-channel conformations with the cytoplasmic-side gate shut, despite bound palytoxin. Modulatory nucleotide binding, therefore, apparently favors cytoplasmic-side gate opening by weakening the cytoplasmic N-, P-, and A-domain interactions that stabilize gate closure, but modulation fails when BeFx strengthens those interactions. [HL36783]

Zwitterionic reagents for labeling, cross-linking and improving the performance of chemiluminescent immunoassays

Improving reagent performance in immunoassays both to enhance assay sensitivity and to minimize interference are ongoing challenges in clinical diagnostics. We describe herein the syntheses of a new class of hydrophilic reagents containing sulfobetaine zwitterions and their applications. These zwitterionic reagents are potentially useful for improving the properties of immunoassay reagents. We demonstrate for the first time that zwitterion labeling is a general and viable strategy for reducing the non-specific binding of proteins to microparticles and, to improve the aqueous solubility of hydrophobic peptides. We also describe the synthesis of zwitterionic cross-linking reagents and demonstrate their utility for peptide conjugation. In automated, chemiluminescent immunoassays, improved assay performance was observed for a hydrophobic, small analyte (theophylline) using an acridinium ester conjugate with a zwitterionic sulfobetaine linker compared to a hexa(ethylene)glycol linker. Sandwich assay performance for a large analyte (thyroid stimulating hormone) was similar for the two acridinium ester labels. These results indicate that zwitterions are complementary to poly(ethylene)glycol in improving the aqueous solubility and reducing the non-specific binding of chemiluminescent acridinium ester conjugates.

Mutational and Cysteine Scanning Analysis of the Glucagon Receptor N-terminal Domain

The glucagon receptor belongs to the B family of G-protein coupled receptors. Little structural information is available about this receptor and its association with glucagon. We used the substituted cysteine accessibility method and three-dimensional molecular modeling based on the gastrointestinal insulinotropic peptide and glucagon-like peptide 1 receptor structures to study the N-terminal domain of this receptor, a central element for ligand binding and specificity. Our results showed that Asp(63), Arg(116), and Lys(98) are essential for the receptor structure and/or ligand binding because mutations of these three residues completely disrupted or markedly impaired the receptor function. In agreement with these data, our models revealed that Asp(63) and Arg(116) form a salt bridge, whereas Lys(98) is engaged in cation-π interactions with the conserved tryptophans 68 and 106. The native receptor could not be labeled by hydrophilic cysteine biotinylation reagents, but treatment of intact cells with [2-(trimethylammonium)ethyl]methanethiosulfonate increased the glucagon binding site density. This result suggested that an unidentified protein with at least one free cysteine associated with the receptor prevented glucagon recognition and that [2-(trimethylammonium)ethyl]methanethiosulfonate treatment relieved this inhibition. The substituted cysteine accessibility method was also performed on 15 residues selected using the three-dimensional models. Several receptor mutants, despite a relatively high predicted cysteine accessibility, could not be labeled by specific reagents. The three-dimensional models show that these mutated residues are located on one face of the protein. This could be part of the interface between the receptor and the unidentified inhibitory protein, making these residues inaccessible to biotinylation compounds.

Residues Gating the Periplasmic Pathway of LacY

X-ray crystal structures of LacY (lactose permease of Escherichia coli) exhibit a large cytoplasmic cavity containing the residues involved in sugar binding and H+ translocation at the apex and a tightly packed side facing the periplasm. However, biochemical and biophysical evidence provide a strong indication that a hydrophilic pathway opens on the external surface of LacY with closing of the cytoplasmic side upon sugar binding. Thus, an alternating-access mechanism in which sugar- and H+-binding sites at the approximate middle of the molecule are alternatively exposed to either side of the membrane is likely to underlie LacY-catalyzed sugar/H+ symport. To further investigate periplasmic opening, we replaced paired residues on the tightly packed periplasmic side of LacY with Cys, and the effect of cross-linking was studied by testing the accessibility/reactivity of Cys148 with the elongated (∼29 Å), impermeant hydrophilic reagent maleimide-PEG2-biotin. When the paired-Cys mutant Ile40→Cys/Asn245→Cys containing native Cys148 is oxidized to form a disulfide bond, the reactivity of Cys148 is markedly inhibited. Moreover, the reactivity of Cys148 in this mutant increases with the length of the cross-linking agent. In contrast, maleimide-PEG2-biotin reactivity of Cys148 is unaffected by oxidation of two other paired-Cys mutants at the mouth of the periplasmic cavity. The data indicate that residues Ile40 and Asn245 play a primary role in gating the periplasmic cavity and provide further support for the alternating-access model.

Biomimetic Synthesis of Calcium Carbonate Polymorphs Using the Lamellar Lyotropic Liquid Crystalline Systems of Calcium Dodecyl Sulfate

Lyotropic liquid crystalline phases have immediate relevance in biology because of the prevalence of organized lipid structures in living systems. The incorporation of hydrophilic reagents in water domains with well-defined nanoscale geometry favors the construction of nanostructured materials of inorganics, and/or the inorganic−organic hybrids. In this paper, the lamellar mesophases composed of calcium dodecyl sulfate (CDS), n-pentanol, and ammonia (5 wt% NH3·H2O in water) were constructed and used as the precipitation media of calcium carbonate (CaCO3). Under the atmosphere of carbon dioxide gas, the occurring solid−liquid−gas reaction resulted only in rhombohedral calcite particles at the beginning, then ultimately in layer-cake aggregates of calcite and stick-bundle aggregates of aragonite. Furthermore, the aragonite content increased with the proceeding time interval of the three-phase reaction, indicating a special crystallization habit of CaCO3 in the lamellar mesophases of CDS. Aside from these, t...

Structure of biodiesel based bicontinuous microemulsions for environmentally compatible decontamination: A small angle neutron scattering and freeze fracture electron microscopy study

Most toxic industrial chemicals and chemical warfare agents are hydrophobic and can only be solubilized in organic solvents. However, most reagents employed for the degradation of these toxic compounds can only be dissolved in water. Hence, microemulsions are auspicious media for the decontamination of a variety of chemical warfare agents and pesticides. They allow for the solubilization of both the lipophilic toxics and the hydrophilic reagent. Alkyl oligoglucosides and plant derived solvents like rapeseed methyl ester enable the formulation of environmentally compatible bicontinuous microemulsions. In the present article the phase behavior of such a microemulsion is studied and the bicontinuous phase is identified. Small angle neutron scattering (SANS) and freeze fracture electron microscopy (FFEM) measurements are used to characterize the structure of the bicontinuous phase and allow for an estimation of the total internal interface. Moreover, also the influence of the co-surfactant (1-pentanol) on the structural parameters of the bicontinuous phase is studied with SANS.

Reactivity in Micelles - Are We Really Able to Design Micellar Catalysts?

Coordination of lipophilic alkyl pyridin-2-yl ketoximes 1 to Ni2+ ions, reduction of lipophilic 3-alkoxyacetophenones 2 with sodium borohydride, and alkaline hydrolysis of 4-nitrophenyl diphenyl phosphate (PNPDPP) were employed as probes in the investigation which factors may influence the reactivity of organic compounds in micellar systems. In all these reactions, a lipophilic substrate solubilized in micellar core was attacked by a hydrophilic reagent from the bulk aqueous phase. To evaluate the contribution of electrostatic interactions between the micellar surface charge and the reagent to the observed reactivity, we combined reactions involving the reagents with opposite polarity (Ni2+ cations and borohydride or hydroxide anions) with positively charged micelles of hexadecyltrimethylammonium chloride (CTAC) or bromide (CTAB) and negatively charged micelles of sodium dodecyl sulfate (SDS). Non-ionic micelles (Triton X-100 or Brij 35) served as a reference. The results of the kinetic studies give evidence that each of the investigated systems has unique properties going in particular aspects beyond the scope of the generally accepted concepts of reactivity in micelles.

Polymeric Sorbents for Virucide Agents in Blood Treatments

Hydrophilic polystyrene (PS) type resins displaying excellent adsorption capacity for aromatic molecules in water were obtained by chemical modification of mesoporous cross-linked PS-type resins with a variety of hydrophilic reagents. The vinyl groups available on the surface of the resin beads were utilized as reactive sites for free radical grafting of hydrophilic moieties. Qualitative and semi-quantitative assessments of the functionalization degree were performed by Fourier transformed infrared (FTIR) spectroscopic analysis. Wetting and water uptake tests confirmed the occurrence of functionalization. The removal of an acridine virucide agent using the modified PS resins was tested under physiological conditions at pH 7.4. The virucide uptake capacity of the modified resins was in some cases almost quantitative. Antipyrogen treatment at 180°C for 8 h did not significantly affect the resin hydrophilicity, although a slight decrease in the adsorption capacity was detected. These modified resins were evaluated in hemofiltration procedures as virucide sorbents during the antiviral stage.

Use of Biotin Derivatives to Probe Conformational Changes in Proteins

Sulfosuccinimidyl-6-(biotinamido) hexanoate and derivatives thereof covalently bind to the epsilon-amino group of lysine residues. Our observation that access of the biotin derivative to specific lysine residues depends on conformational properties of the entire polypeptide chain prompted us to investigate whether differential biotinylation patterns of a protein can be used as indicators for conformational changes. Bovine serum albumin is a soluble protein with characteristic unfolding kinetics upon exposure to high temperature. First, we show that biotinylation patterns of proteins are highly reproducible. Second, we demonstrate by mass spectrometry and tandem mass spectrometry that unfolding of the protein correlates with the accessibility of the biotin derivative to specific lysine residues. We have applied this experimental strategy to the analysis of a cell-surface protein, viz. the human band 3 anion exchanger of erythrocytes infected with the malaria parasite Plasmodium falciparum. We found that Lys(826) in a highly flexible loop can be biotinylated in non-infected (but not infected) erythrocytes, confirming earlier observations (Winograd, E., and Sherman, I. W. (2004) Mol. Biochem. Parasitol. 138, 83-87) based on epitope-specific monoclonal antibodies suggesting that this region undergoes a conformational change upon infection.

Polymeric Nanoreactors for Hydrophilic Reagents Synthesized by Interfacial Polycondensation on Miniemulsion Droplets

We describe a versatile method to obtain functional hollow nanoreactors with a hydrophilic liquid core. The synthesis of hollow polyurea, polythiourea, and polyurethane nanocapsules was performed by interfacial polycondensation or cross-linking reactions in inverse miniemulsion. The miniemulsions were built upon emulsification of a solution of amines or alcohols in a polar solvent with cyclohexane as the nonpolar continuous phase. The addition of suitable hydrophobic diisocyanate or diisothiocyanate monomers to the continuous phase allows the polycondensation or the cross-linking reactions to occur at the interface of the droplets. The wall thickness of the capsules can be directly tuned by the quantity of the reactants. The nature of the monomers and the continuous phase are the critical factors for the formation of the hollow capsules, which is explained by the interfacial properties of the system. The resulting polymer nanocapsules could be subsequently dispersed in water. The capsules were found to be...

Identification of Oxidized Methionine Sites in Erythrocyte Membrane Protein by Liquid Chromatography/Electrospray Ionization Mass Spectrometry Peptide Mapping

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.

The effect of chemical modification of amino acid side‐chains on collagen degradation by enzymes

In this study, the effects of specific chemical modifications of amino acid side-chains on the in vitro enzyme degradation of type I collagen was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine and methylglyoxal was used to modify arginine. Lysine residues were modified using glycidol, a small hydrophilic reagent or n-butylglycidylether, a larger hydrophobic reagent. Amino acid analysis, swelling measurements, in vitro enzyme degradation analyses (using either collagenase, trypsin, acetyltrypsin, or cathepsin B), and gel chromatography were used to determine the effects of each chemical modification on purified type I collagen. Collagen solubilization by enzymes depended upon the size and chemistry of epoxides used to modify lysine residues. Modification of lysine residues by glycidol and arginine modification by methylglyoxal together significantly reduced collagen solubilization by acetyltrypsin and collagenase, whereas increased collagen solubilization was observed for all enzymes after lysine modification with n-butylglycidylether combined with arginine modification by methylglyoxal. Gel chromatographic analyses of collagen fragments solubilized by acetyltrypsin from type I collagen revealed that both the extent of solubilization and sites of cleavage were altered after lysine and arginine modification. In contrast, lysine and arginine modification only altered the amount of collagen solubilized by collagenase and had no effect on the amount collagen solubilized by cathepsin B. The ability to modulate the enzyme degradation of collagen-based materials as demonstrated in this study may facilitate the design of novel scaffolds for tissue regeneration or collagen-based drug/protein/gene delivery systems.

Exploring the Role of a Unique Carboxyl Residue in EmrE by Mass Spectrometry

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.

Accessibility of Cysteines in the Native Bovine Rod cGMP-Gated Channel

Cyclic nucleotide-gated channels of photoreceptors and olfactory sensory neurons are tetramers consisting of A and B subunits. Here, the accessibility of the cysteines of the bovine rod cyclic nucleotide-gated channel is examined as a function of ligand binding. N-Ethylmaleimide-modified cysteines of both subunits were identified by mass spectrometry after trypsin digestion. In the absence of ligand, the intracellular carboxy-terminal cysteines of both subunits were accessible to N-ethylmaleimide. Activation of the channel abolished the accessibility of Cys505 of the A subunit and Cys1104 of the B subunit, with both being conserved cysteines of the cyclic nucleotide-binding sites. The cysteine of the pore loop of the B subunit was also found to be modified by this reagent in the absence of ligand. The total number of accessible cysteines of each subunit was determined by mass shifting upon modification with polyethylene glycol maleimide. In the absence of cyclic nucleotides, this hydrophilic reagent only weakly labeled cysteines of the A subunit but readily labeled at least three cysteines of the B subunit. Ligand binding exposed two cysteines of the A subunit and one cysteine of the B subunit to chemical modification. Double-modification experiments suggest that some of these cysteines are in or close to membrane-spanning domains. However, these cysteines could not yet be identified. Together, the cysteine accessibility of the native rod cyclic nucleotide-gated channel varies markedly upon ligand binding, thus indicating major structural rearrangements, which are of functional importance for channel activation.

Removal of viruscide agents by using styrenic resins.

Strong and weak anionic polystyrene/divinylbenzene ion exchange resins were investigated both as iodophores and as iodine/iodide ion removal agents in blood disinfection applications. Resin-iodine complexes were prepared, but there was no significant iodine release observed in either distilled water or isotonic saline solution. However, all ion-exchange resin were able to remove almost quantitatively both iodine and iodide ions from water solutions. Cross-linked styrene/divinylbenzene resins are excellent polyaromatic viruscide adsorbents, although their hydrophobicity is responsible for poor wettability in physiologi-cal fluids. Surface modification with hydrophilic reagents appeared a promising strategy to overcome this drawback. A Merrifield-type chloromethylated resin and a highly cross-linked mesoporous resin (Lewatit 1064) with a large surface area and content of unreacted vinyl groups were selected as starting materials. The Merrifield resin was modified by the reaction of the pendant chloromethyl groups with triethyleneglycol, tetraethyleneglycol and â -cyclodextrin. The conversion of Lewatit double bonds into hydrophilic moieties was attempted by the radical grafting of N-vinyl-2-pyrrolidinone (NVP), maleic anhydride (MAn), 2-hydroxyethyl methacrylate, acrylamide (AAm) and different poly(ethyleneglycol) (PEG) methacrylates. The addition of 2-mer-captoethanol (2ME) and epoxidation were also investigated. The modified Merrifield resins demonstrated very low efficiency in acridine viruscide uptake, in spite of the large increase in both wettability and water uptake. On the other hand, all modified Lewatit samples removed rapidly and almost quantitatively the viruscide from aqueous solutions, although only a few samples resulted in being very hydrophilic. In all cases, hydrophilicity and viruscide adsorbing capacity were maintained after heating at 180 degrees C to simulate pyrogen elimination.

Chloro(2,2':6',2"-terpyridine) platinum inhibition of the renal Na+,K+-ATPase.

Chloro(2,2':6',2"-terpyridine) platinum, a bulky, hydrophilic reagent, inhibited the renal sodium pump with a single exponential time course. K(+) increased the rate constant of the reaction by about twofold; the K(+) concentration dependence was monotonic, with a half-maximal effect observed at 1 mM, consistent with K(+) acting at a transport site. Na(+), Mg(2+), eosin, and vanadate did not significantly alter the rate of reaction. The results of proteolysis and mass spectrometer analysis were consistent with terpyridine platinum labeling of Cys452, Cys456, or Cys457. Because phenylarsine oxide reacts with vicinal cysteines and did not prevent terpyridine platinum modification, terpyridine platinum most likely modifies Cys452. This modification prevents ADP binding; interestingly, the analogous residue in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is on the exterior of the nucleotide-binding pocket. Thus it appears that the terpyridine platinum residue is more accessible in the presence of K(+) than in its absence and that terpyridine platinum modification prevents nucleotide binding.

Identification of Surface Proteins of Helicobacter pylori by Selective Biotinylation, Affinity Purification, and Two-dimensional Gel Electrophoresis

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

Cysteine Substitutions for Individual Residues in Helix VI of the Melibiose Carrier of Escherichia coli

The melibiose carrier of Escherichia coli is a cytoplasmic membrane protein that mediates the cotransport of galactosides with H(+), Na(+), or Li(+). In this study we used cysteine-scanning mutagenesis to try to gain information about the position of transmembrane helix VI in the three-dimensional structure of the melibiose carrier. We constructed 23 individual cysteine substitutions in helix VI and an adjacent loop of the carrier. The resulting melibiose carriers retained 22-100% of their ability to transport melibiose. We tested the effect of the hydrophilic sulfhydryl reagent p-chloromercuri-benzenesulfonic acid (PCMBS) on the cysteine-substitution mutants and we found that there was no inhibition of melibiose transport in any of the mutants. We suggest that helix VI is imbedded in phospholipid and does not face the aqueous channel through which melibiose passes.