Hydrophilic Cavity Research Articles

Packing of the Extracellular Domain Hydrophobic Core is Evolutionarily Optimized to Facilitate Pentameric Ligand-Gated Ion Channel Activation

Protein function depends on protein dynamics as well as structure. Local loose packing of hydrophobic cores is not infrequent in proteins, as the enhanced flexibility likely contributes to their biological function. The crystal structure of the extracellular domain of the nicotinic acetylcholine receptor (nAChR) α1 subunit revealed a hydrophilic water-filled cavity formed by Thr-52 and Ser-126 buried in the hydrophobic core of the protein. Mutation of these residues to bulky hydrophobic amino acids substantially reduced acetylcholine activated channel current suggesting loose-packing of the β-sandwich core is important for nAChR function. Intriguingly, structure-based sequence alignment suggests the presence of loose packing of the hydrophobic β-sandwich core in other pentameric ligand-gated ion channels (pLGICs), whereas tight packing is observed in the crystal structures of the nonchannel homolog, acetylcholine binding protein (AChBP). Here, we examined the generality and importance of this loose packing for pLGIC function using experimental and computational approaches. Mutating aligned residues in the related heteropentameric GABA-A receptor disrupted GABA-mediated currents. Mutations in the β2 subunit had the largest effects suggesting distinct requirements for subunit flexibility in receptor activation. Using FoldX, we examined the energetic cost of mutating residues in the hydrophobic core on protein folding in AChBP, prokaryotic and eukaryotic pLGICs as a measure of protein stability. Interestingly, a loss in protein stability appears correlated to the ability of pLGICs to rapidly switch from closed to open channel states in the presence of ligand. Overall, we suggest that loose packing of the hydrophobic core likely developed as an evolutionary strategy aimed to optimize the specialized allosteric mechanisms of pLGICs.

Probing of the rates of alternating access in LacY with Trp fluorescence.

Sugar/H(+) symport by lactose permease (LacY) utilizes an alternating access mechanism in which sugar and H(+) binding sites in the middle of the molecule are alternatively exposed to either side of the membrane by sequential opening and closing of inward- and outward-facing hydrophilic cavities. Here, we introduce Trp residues on either side of LacY where they are predicted to be in close proximity to side chains of natural Trp quenchers in either the inward- or outward-facing conformers. In the inward-facing conformer, LacY is tightly packed on the periplasmic side, and Trp residues placed at positions 245 (helix VII) or 378 (helix XII) are in close contact with His-35 (helix I) or Lys-42 (helix II), respectively. Sugar binding leads to unquenching of Trp fluorescence in both mutants, a finding clearly consistent with opening of the periplasmic cavity. The pH dependence of Trp-245 unquenching exhibits a pK(a) of 8, typical for a His side chain interacting with an aromatic group. As estimated from stopped-flow studies, the rate of sugar-induced opening is approximately 100 s(-1). On the cytoplasmic side, Phe-140 (helix V) and Phe-334 (helix X) are located on opposite sides of a wide-open hydrophilic cavity. In precisely the opposite fashion from the periplasmic side, mutant Phe-140-->Trp/Phe-334-->His exhibits sugar-induced Trp quenching. Again, quenching is pH dependent (pK(a) = 8), but remarkably, the rate of sugar-induced quenching is only approximately 0.4 s(-1). The results provide yet another strong, independent line of evidence for the alternating access mechanism and demonstrate that the methodology described provides a sensitive probe to measure rates of conformational change in membrane transport proteins.

Properties of a LacY Efflux Mutant

Crystal structures of lactose permease from Escherichia coli (LacY) exhibit two six-helix bundles with 2-fold pseudosymmetry separated by a large hydrophilic cavity. The cavity is open only on the cytoplasmic side and contains the side chains important for both sugar and H(+) binding at the apex in the middle of the protein; the periplasmic side is tightly closed. A plethora of biochemical and biophysical data strongly support an alternating access mechanism in which both the sugar- and H(+)-binding sites are exposed alternatively to either side of the membrane by reciprocal opening and closing of cytoplasmic and periplasmic cavities. Here we describe a unique mutation that results in an increase in sugar efflux. Asp240 (helix VII), which interacts with Lys319 (helix X), also comprises part of a salt-bridge/H-bond network that is critically involved in the mechanism of sugar/H(+) symport. The mutant, which contains Glu in place of Asp240, exhibits a marked decrease in active lactose transport and an enhanced rate of downhill lactose/H(+) efflux. Transport is increased to normal levels when the sugar concentration is increased 10-fold, consistent with the decrease in sugar affinity observed for this mutant. Taken as a whole, the results suggest that the primary defect induced by the mutation may involve a decrease in affinity for H(+).

A 3D structure model of the melibiose permease of Escherichia coli represents a distinctive fold for Na + symporters

The melibiose permease of Escherichia coli (MelB) catalyzes the coupled stoichiometric symport of a galactoside with a cation (either Na(+), Li(+), or H(+)), using free energy from the downhill translocation of one cosubstrate to catalyze the accumulation of the other. Here, we present a 3D structure model of MelB threaded through a crystal structure of the lactose permease of E. coli (LacY), manually adjusted, and energetically minimized. The model contains 442 consecutive residues ( approximately 94% of the polypeptide), including all 12 transmembrane helices and connecting loops, with no steric clashes and superimposes well with the template structure. The electrostatic surface potential calculated from the model is typical for a membrane protein and exhibits a characteristic ring of positive charges around the periphery of the cytoplasmic side. The 3D model indicates that MelB consists of two pseudosymmetrical 6-helix bundles lining an internal hydrophilic cavity, which faces the cytoplasmic side of the membrane. Both sugar and cation binding sites are proposed to lie within the internal cavity. The model is consistent with numerous previous mutational, biochemical/biophysical characterizations as well as low-resolution structural data. Thus, an alternating access mechanism with sequential binding is discussed. The proposed overall fold of MelB is different from the available crystal structures of other Na(+)-coupled transporters, suggesting a distinctive fold for Na(+) symporters.

Invertible Amphiphilic Molecular Pockets Made of Cholic Acid

A trimer of cholic acid was prepared via a simple isocyanate linkage. Fluorescence and UV-vis spectroscopic studies showed that the same cholane trimer can form either a hydrophobic cavity in aqueous solutions or a hydrophilic cavity in an apolar solvent using pyrene and a derivative of pyrene as probes. The dynamic inversion of the cavities was demonstrated for the first time by a simple change of the polarity of the solvent media by adjusting from an aqueous buffer to tetrahydrofuran and vice versa. The NMR study of a dimer model compound of cholic acid indicated a restricted conformation exist in the solvent mixture of chloroform and methanol.

Chaperonin-mediated protein folding: using a central cavity to kinetically assist polypeptide chain folding

The chaperonin ring assembly GroEL provides kinetic assistance to protein folding in the cell by binding non-native protein in the hydrophobic central cavity of an open ring and subsequently, upon binding ATP and the co-chaperonin GroES to the same ring, releasing polypeptide into a now hydrophilic encapsulated cavity where productive folding occurs in isolation. The fate of polypeptide during binding, encapsulation, and folding in the chamber has been the subject of recent experimental studies and is reviewed and considered here. We conclude that GroEL, in general, behaves passively with respect to its substrate proteins during these steps. While binding appears to be able to rescue non-native polypeptides from kinetic traps, such rescue is most likely exerted at the level of maximizing hydrophobic contact, effecting alteration of the topology of weakly structured states. Encapsulation does not appear to involve 'forced unfolding', and if anything, polypeptide topology is compacted during this step. Finally, chamber-mediated folding appears to resemble folding in solution, except that major kinetic complications of multimolecular association are prevented.

Novel surface modified molecularly imprinted polymer using acryloyl-β-cyclodextrin and acrylamide as monomers for selective recognition of lysozyme in aqueous solution

A novel protein imprinted polymer for selective recognition of lysozyme was obtained. Acryloyl-β-cyclodextrin, which offered hydrophilic exterior and hydrophobic cavity that were allowed to self-assemble with the template protein through hydrogen interaction and hydrophobic interaction, was synthesized and used as the functional monomer. Polymerization was carried out in the presence of acrylamide as an assistant monomer, which resulted in a new type of protein imprinted polymer. Langmuir adsorption model was employed to describe the isotherms, and maximum adsorption capacity was evaluated. The performance of such imprinted polymer was further demonstrated by high-performance liquid chromatography, and the results showed that the column packed with the lysozyme imprinted silica beads could effectively separate lysozyme from the mixture of lysozyme–cytochrome c, lysozyme–bovine serum albumin, lysozyme–avidin or lysozyme–methylated bovine serum albumin, which showed its high selectivity.

Cu(ii) complexation with an acridine-containing macrocycle. Assembly of water cluster chains within the cavity of tetranuclear metallomacrocycles

The synthesis and characterisation of a new macrocyclic compound (L), composed of a pentamine chain linking the 2,7 positions of an acridine moiety, is reported. Cu(ii) complexation was studied by means of potentiometric, UV-vis and EPR measurements in aqueous solutions. This study reveals that the ligand forms a stable tetranuclear complex with an overall [Cu(4)L(2)(OH)(4)](4+) stoichiometry in aqueous solution. The crystal structure of the [Cu(8)L(4)(micro-OH)(8)(micro-NO(3))(3)](NO(3))(5).32H(2)O complex, isolated from neutral aqueous solution, shows that it is formed by tetranuclear clusters [Cu(4)L(2)(micro-OH)(4)(micro-NO(3))(x)]((4-x)+) (x= 1 or 2), composed of two [Cu(2)L](4+) macrocyclic units linked together by four bridging hydroxide and nitrate anions to give an overall metallomacrocyclic structure. Magnetic measurements shows that the hydroxide-bridged Cu(ii) ions are ferromagnatically coupled. Inspection of the crystal packing shows that couples of metallomacrocycles are paired by pi-stacking interactions between acridine moieties, giving rise to an internal hydrophilic cavity were hexameric or pentameric water clusters are enclosed. The coupled metallomacrocycles assume a columnar disposition growing along the a axis, giving rise to channels passing through the cavities of the metallomacrocycles; these channels are filled by chains of 5- or 6-membered water clusters linked together by [NO(3).H(2)O](2) units.

β-Cyclodextrin containing Matrimid ® sub-nanocomposite membranes for pervaporation application

Molecular-level mixed matrix membranes made of Matrimid ®/β-cyclodextrin (β-CD) sub-nanocomposites for dehydrating isopropanol by pervaporation have been fabricated and studied. The separation efficiency of such membranes containing ∼2 to 10 wt.% of β-CD is superior over the neat Matrimid ® membrane in terms of both flux and separation factor. However, phase separation due to β-CD agglomeration occurs with an increase in β-CD loading. Asymmetry in both morphology and surface property has also been observed. The separation properties depend upon the membrane mounting mode (i.e., using air-side or glass-side of the membrane against the feed during pervaporation tests). The flux and separation factor of both modes, nevertheless, display the same increase-and-decrease dependence on β-CD loading. The unique characteristics of β-CD such as hydrophilic exterior and interior cavity and the interactions between β-CD molecules and the polymer matrix may create additional interface channels for water diffusion and provide size discrimination and chain rigidification for water/isopropanol selection. As a result, compared to mixed matrix membranes incorporated MgO or zeolite nanoparticles, embedding a low percentage of β-CD into the Matrimid ® leads to the enhanced and superior separation efficiency. On the other hand, a higher loading of β-CD deteriorates separation performance due to the formation of a non-permeable β-CD network. The proposed Matrimid ®/β-CD mixed matrix materials may open new perspective for the development of mixed-matrix hollow-fiber membranes because of the sub-nano-sized β-CD and its unique molecular-level mixing characteristics.

Combined Crystallographic and Solution Molecular Dynamics Study of Allosteric Effects in Ester and Ketone p-tert-Butylcalix[4]arene Derivatives and Their Complexes with Acetonitrile, Cd(II), and Pb(II)

We describe here a procedure to bridge the gap in the field of calixarene physicochemistry between solid-state atomic-resolution structural information and the liquid-state low-resolution thermodynamics and spectroscopic data. We use MD simulations to study the kinetics and energetics involved in the complexation of lower rim calix[4]arene derivatives (L), containing bidentate ester (1) and ketone (2) pendant groups, with acetonitrile molecule (MeCN) and Cd(2+) and Pb(2+) ions (M(2+)) in acetonitrile solution. On one hand, we found that the prior inclusion of MeCN into the calix to form a L(MeCN) adduct has only a weak effect in preorganizing the hydrophilic cavity toward metal ion binding. On the other hand, the strong ion-hydrophilic cavity interaction produces a wide open calix which enhances the binding of one MeCN molecule (allosteric effect) to stabilize the whole (M(2+)) L(MeCN) bifunctional complex. We reach two major conclusions: (i) the MD results for the (M(2+)) 1(MeCN) binding are in close agreement with the "endo", fully encapsulated, metal complex found by X-ray diffraction and in vacuo MD calculations, and (ii) the MD structure for the more flexible 2 ligand, however, differs from the also endo solid-state molecule. In fact, it shows strong solvation effects at the calixarene lower bore by competing MeCN molecules that share the metal coordination sphere with the four CO oxygens of an "exo" (M(2+)) 2(MeCN) complex.

Charge-Transfer Dynamics in Cyano-BridgedMA–Fe System (MA=Mn, Fe, and Co)

Charge-transfer (CT) dynamics has been investigated for Prussian blue-type M A –Fe cyanides ( M A =Mn, Fe, and Co) grown in hydrophilic cavities of a Nafion 117 film. In all the compounds, we observed the suppression of the original CT absorption between neighboring transition metals. We found that the spectral profile of the slow component for the Fe compound is similar to that for the Co compound, reflecting the photo created d 6 state at the M A site.

Function of Transmembrane Domain IX in the Na+/Proline Transporter PutP

Selected residues of transmembrane domain (TM) IX were previously shown to play key roles in ligand binding and transport in members of the Na+/solute symporter family. Using the Na+/proline transporter PutP as a model, a complete Cys scanning mutagenesis of TM IX (positions 324 to 351) was performed here to further investigate the functional significance of the domain. G328, S332, Q345, and L346 were newly identified as important for Na+-coupled proline uptake. Placement of Cys at one of these positions altered Km(pro) (S332C and L346C, 3- and 21-fold decreased, respectively; Q345C, 38-fold increased), K0.5(Na+) (S332C, 13-fold decreased; Q345C, 19-fold increased), and/or Vmax [G328C, S332C, Q345C, and L346C, 3-, 22-, 2-, and 8-fold decreased compared to PutP(wild type), respectively]. Membrane-permeant N-ethylmaleimide inhibited proline uptake into cells containing PutP with Cys at distinct positions in the middle (T341C) and cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) and had little or no effect on all other single Cys PutP variants. The inhibition pattern was in agreement with the pattern of labeling with fluorescein-5-maleimide. In addition, Cys placed into the cytoplasmic half of TM IX (C344, L347C, V348C, and S351C) was protected from fluorescein-5-maleimide labeling by proline while Na+ alone had no effect. Membrane-impermeant methanethiosulfonate ethyltrimethylammonium modified Cys in the middle (A337C and T341C) and periplasmic half (L331C) but not in the cytoplasmic half of TM IX in intact cells. Furthermore, Cys at the latter positions was partially protected by Na+ but not by proline. Based on these results, a model is discussed according to which residues of TM IX participate in the formation of ligand-sensitive, hydrophilic cavities in the protein that may reconstitute part of the Na+ and/or proline translocation pathway of PutP.

Transmembrane Domain 9 of Presenilin Determines the Dynamic Conformation of the Catalytic Site of γ-Secretase

One of the most prominent drug targets for the treatment of Alzheimer disease is gamma-secretase, a multi-protein complex responsible for the generation of the amyloid-beta peptide. The catalytic core of the complex lies on presenilin, a multi-spanning membrane protease, the activity of which depends on two aspartate residues located in transmembrane domains 6 and 7. We have recently shown by cysteine-scanning mutagenesis that these aspartates are facing a water-filled cavity in the lipid bilayer, demonstrating how proteolytic cleavage of the substrates can be taking place within the membrane. Here, we demonstrate that transmembrane domain 9 and hydrophobic domain VII in the large cytoplasmic loop of presenilin are dynamic structural parts of this cavity. Hydrophobic domain VII is associated with transmembrane domain 7 in the membrane, probably facilitating the entrance of water molecules in the catalytic site. Transmembrane domain 9, on the other hand, exhibits a highly flexible structure, potentially involved in the transport of substrates to the catalytic site, as well as in the binding of gamma-secretase inhibitors. The conserved proline-alanine-leucine motif at the cytoplasmic part of this domain is extremely close to the catalytic Asp257 and is crucial for conformational changes leading to the activation of the catalytic site. We, also, identify a unique mutant in this domain (I437C) that specifically blocks amyloid-beta peptide production without affecting the processing of the physiologically indispensable Notch substrate. Our data are finally combined to propose a model for the architectural organization and activation of the catalytic site of presenilin.

Dynamics of charge-transfer pairs in the cyano-bridgedCo2+−Fe3+transition-metal compound

Dynamics of the charge-transfer (CT) pairs has been investigated for the cyano-bridged ${\mathrm{Co}}^{2+}\text{\ensuremath{-}}{\mathrm{Fe}}^{3+}$ particles grown in a hydrophilic cavities of Nafion 117 film. We decomposed the differential absorption spectra into the fast and slow components. We ascribed the fast and slow components to the Frank-Condon state and the CT $({\mathrm{Co}}^{3+}\text{\ensuremath{-}}{\mathrm{Fe}}^{2+})$ pairs with lattice relaxation, respectively. We found that the lifetime ${\ensuremath{\tau}}_{\mathrm{CT}}$ of the pairs at $10\phantom{\rule{0.3em}{0ex}}\mathrm{K}$ increases with the absorbed photon density $n$ from ${\ensuremath{\tau}}_{\mathrm{CT}}\ensuremath{\approx}2\phantom{\rule{0.3em}{0ex}}\mathrm{ns}\phantom{\rule{0.3em}{0ex}}\text{to}\phantom{\rule{0.3em}{0ex}}\ensuremath{\approx}6\phantom{\rule{0.3em}{0ex}}\mathrm{ns}$, reflecting the attractive interaction between the pairs.

Structural determination of wild-type lactose permease

Here we describe an x-ray structure of wild-type lactose permease (LacY) from Escherichia coli determined by manipulating phospholipid content during crystallization. The structure exhibits the same global fold as the previous x-ray structures of a mutant that binds sugar but cannot catalyze translocation across the membrane. LacY is organized into two six-helix bundles with twofold pseudosymmetry separated by a large interior hydrophilic cavity open only to the cytoplasmic side and containing the side chains important for sugar and H(+) binding. To initiate transport, binding of sugar and/or an H(+) electrochemical gradient increases the probability of opening on the periplasmic side. Because the inward-facing conformation represents the lowest free-energy state, the rate-limiting step for transport may be the conformational change leading to the outward-facing conformation.

Interactions of theophylline with cyclodextrins in water

Theophylline (1,3-dimethylxanthine, Figure 1), a poorly water soluble purine alkaloid, is widely used in medicine.1,2 The use of pure theophylline is limited by its very fast absorption and conversion into inactive metabolites. Moreover, theophylline in high doses causes unwanted side effects.3 To remove these disadvantages, coated controlled-release theophylline tablet formulations have been obtained in recent years. Acrylic and ion-exchange resins, paraffin, ethyl cellulose4–6 and cyclodextrins (CDs)7–9 were proposed as coating and encapsulating materials. The use of CDs as solubilising and stabilising agents is based on their ability to form inclusion complexes with organic substrates.10 The most commonly used α-, βand γ-cyclodextrins consist of six to eight glucopyranose units, respectively, and possess hydrophilic exterior and hydrophobic internal cavity. Placing inside the CD molecular cavity the substrate can considerably improve physico-chemical and pharmacological properties (solubility, stability, bioactivity, etc.).

Enzymatic analysis of a rhomboid intramembrane protease implicates transmembrane helix 5 as the lateral substrate gate

Intramembrane proteolysis is a core regulatory mechanism of cells that raises a biochemical paradox of how hydrolysis of peptide bonds is accomplished within the normally hydrophobic environment of the membrane. Recent high-resolution crystal structures have revealed that rhomboid proteases contain a catalytic serine recessed into the plane of the membrane, within a hydrophilic cavity that opens to the extracellular face, but protected laterally from membrane lipids by a ring of transmembrane segments. This architecture poses questions about how substrates enter the internal active site laterally from membrane lipid. Because structures are static glimpses of a dynamic enzyme, we have taken a structure-function approach analyzing >40 engineered variants to identify the gating mechanism used by rhomboid proteases. Importantly, our analyses were conducted with a substrate that we show is cleaved at two intramembrane sites within the previously defined Spitz substrate motif. Engineered mutants in the L1 loop and active-site region of the GlpG rhomboid protease suggest an important structural, rather than dynamic, gating function for the L1 loop that was first proposed to be the substrate gate. Conversely, three classes of mutations that promote transmembrane helix 5 displacement away from the protease core dramatically enhanced enzyme activity 4- to 10-fold. Our functional analyses have identified transmembrane helix 5 movement to gate lateral substrate entry as a rate-limiting step in intramembrane proteolysis. Moreover, our mutagenesis also underscores the importance of other residue interactions within the enzyme that warrant further scrutiny.

Topologies of a Substrate Protein Bound to the Chaperonin GroEL

SummaryThe chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.

Perturbed ATPase activity and not “close confinement” of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of "dwell time" for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the approximately 10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of "close confinement" on folding in the cis cavity.

Nanocrystalline zinc oxide in perfluorinated ionomer membranes: Preparation, characterization, and photocatalytic properties

Crystalline ZnO nanoparticles were synthesized via the templating method by taking advantage of the hydrophilic cavities of the Nafion ionomer membranes. The ZnO-embedded Nafion film was characterized by various methods, including optical absorption, X-ray diffraction and high-resolution transmission electron microscopy. The film was of high optical quality, and exhibited excellent photocatalytic activity toward rhodamine B degradation under UV irradiation. Unlike unprotected bulk ZnO nanocrystals, the ZnO nanocatalysts embedded in Nafion membranes were found to be highly stable against photocorrosion, with hardly any reduction in the activity even after 10 times of repeated reaction cycles. The implications of the results are discussed.