Weak Hydrophobic Interactions Research Articles

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.

Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and weak hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of principle, we demonstrate that the soluble expression of an aggregation-prone positively charged intrabody is modestly enhanced via cis or trans acidification using highly charged peptide tags (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use.

Ablation of the Complementarity-Determining Region H3 Apex of the Anti-HIV-1 Broadly Neutralizing Antibody 2F5 Abrogates Neutralizing Capacity without Affecting Core Epitope Binding

The identification and characterization of broadly neutralizing antibodies (bnAbs) against HIV-1 has formed a major research focus, with the ultimate goal to help in the design of an effective AIDS vaccine. One of these bnAbs, 2F5, has been extensively characterized, and residues at the apex of its unusually long complementarity-determining region (CDR) H3 loop have been shown to be crucial for neutralization. Structural studies, however, have revealed that the (100)TLFGVPI(100F) apex residues of the CDR H3 loop do not interact directly with residues of its core gp41 epitope. In an attempt to gain better insight into the functional role of this element, we have recombinantly expressed native 2F5 Fab and two mutants in which either the apical Phe100B(H) residue was changed to an alanine or the CDR H3 residues (100)TLFGVPI(100F) were replaced by a Ser-Gly dipeptide linker. Isothermal titration calorimetry (ITC) and competitive-binding enzyme-linked immunosorbent assays (ELISAs) rendered strikingly similar affinity constants (K(d) [dissociation constant] of approximately 20 nM) for linear peptide epitope binding by 2F5 Fabs, independent of the presence or absence of the apex residues. Ablation of the CDR H3 apex residues, however, abolished the cell-cell fusion inhibition and pseudovirus neutralization capacities of 2F5 Fab. We report competitive ELISA data that suggest a role of 2F5 CDR H3 apex residues in mediating weak hydrophobic interactions with residues located at the C terminus of the gp41 membrane proximal external region and/or membrane components in the context of core epitope binding. The present data therefore imply an extended 2F5 paratope that includes weak secondary interactions that are crucial for neutralization of Env-mediated fusion.

An estimation of hydrophilic and hydrophobic interaction of aqueous urea, methylurea, dimethylurea and tetramethylurea from density and apparent molal volume at 30.0°C

Density (ρ) for 0.20–1.0 mol kg−1 of urea, 1-methylurea, 1,3-dimethylurea and 1,1,3,3-tetramethylurea solutions have been measured at an interval of 0.2 mol kg−1. Apparent molal volume (V φ, cm3 mol−1) is calculated from ρ values. Primary data were regressed and extrapolated to zero concentration for the limiting density (ρ0) and limiting apparent molal volume values for solute–solvent interactions.–CH3 (methyl) groups of N-methylureas weaken hydrophilic interaction and enhance hydrophobic interaction. The value of ρ0 and reflect the intermolecular forces due to electrostatic charge. The decreasing value of ρ0 and increasing value of with increasing number of–CH3 groups suggest some weak hydrophilic and strong hydrophobic interactions, so that the structure-breaking effect decreases. It was also found that with increasing concentration, the hydrophilic or hydrophobic interactions become stronger.

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.

New cyclodextrin derivative 6- O-(2-hydroxybutyl)-β-cyclodextrin: preparation and its application in molecular binding and recognition

A new soluble cyclodextrin derivative 6- O-(2-hydroxybutyl)-β-cyclodextrin (6-HB-β-CD) was prepared. Its molecular binding and recognition ability were investigated with the comparison of β-cyclodextrin (β-CD), 2- O-(2-hydroxypropyl)-β-cyclodextrin (2-HP-β-CD), 6- O-(2-hydroxypropyl)-β-cyclodextrin (6-HP-β-CD), and 2- O-(2-hydroxybutyl)-β-cyclodextrin (2-HB-β-CD). The relationship between the complex stability constants and the possible structures of inclusion compounds was discussed with the interaction of hosts and guests, including the weak hydrophobic interactions, the size/shape matching, the steric hindrance, and the hydrophilic property.

Reliable Formation of an Unusual and Chiral Two-Dimensional Network Containing Entanglement of the Ligand in the Presence of Different Anions

The coordination polymers of the ligand N,N′-bis(4-pyridyl)suberamide (1a) with Cu(II) were explored in the presence of anions such as bivalent SO42− and SiF62− and univalent NO3−1. It was found that the ligand with Cu(II) ion consistently forms an unusual two-dimensional layer containing (4,4)-topology irrespective of the anions present. In the layer, the alkyl chains are entangled via weak hydrophobic interactions such that the chirality is generated in the layer. The entangled spacers occupy grid cavities with dimensions of 12 × 12 A. These layers are further linked via hydrogen bonds between amide N−H groups and anions (SO42− or SiF62−).

A librational entropy mechanism for elastomers with repeating peptide sequences in helical array

A librational entropy mechanism is proposed for the elastomeric polypentapeptide of elastin which derives from the particular properties of a previously described dynamic β-spiral structure comprised of the repeating pentamer sequence. Using a cutoff energy of 2 kcal/mol residue, it is shown that the number of conformational states accessible to a single pentamer segment without perturbing the rest of the β-spiral is large in the relaxed state but becomes greatly decreased on extension. With use of the Boltzmann relation, it is seen that the effect of identical repeating units in a dynamic β-spiral is to linearly increase the entropy as the chain length increases. This occurs in spite of the valyl and prolyl side chains that significantly limit the states of the polymer and the Pro-Gly sequence which inserts a flexible β-turn. The aggregated states of the dynamic β-spiral with their weak hydrophobic intermolecular interactions that occur at high concentrations of chains and at physiologic temperatures are presented as an effective means of avoiding the entanglements that can limit numbers of accessible states for a concentrated random tangle of chains. Thus, a new mechanism is presented for an entropic elastomer which does not require that the relaxed state be totally random.

Interaction of E1 and E3 components with the core proteins of the human pyruvate dehydrogenase complex

The human (h) pyruvate dehydrogenase complex (hPDC) consists of multiple copies of several components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), E3-binding protein (BP), and specific kinases and phosphatases. Mammalian PDC has a well-organized structure with an icosahedral symmetry of the central E2/BP core to which the other component proteins bind non-covalently. Both hE2 and hBP consist of three well defined domains, namely the lipoyl domain, the subunit-binding domain and the inner domain, connected with flexible linkers. hE1 (α 2β 2) binds to the subunit-binding domain of hE2; whereas hE3 binds to the E3-binding domain of hBP. Among several residues of the C-terminal surface of the hE1β, E1βD289 was found to interact with hE2K276. The C-terminal residue I329 of the hE1β did not participate in binding to hE2. This latter finding shows specificity in the interaction between E1β and E2 in hPDC. The selective binding between hE3 and the E3-binding domain of hBP was investigated using specific mutants. E3R460G and E3340K showed significant reductions in affinity for hBP as determined by surface plasmon resonance. Both residues are involved in the structural organization of the binding site on hE3. Substitution of I157, N137 and R155 of hBP resulted in variable increases in the K D for binding with wild-type hE3, suggesting that the binding results from several weak electrostatic bonds and hydrophobic interactions among residues of hBP with residues at the interface of dimeric hE3. These results provide insight in the mono-specificity of binding of E1 to E2 and E3 to BP in hPDC and showed the differences in the binding of peripheral components (E1 and E3) in human and bacterial PDCs.

Molecular Insight into Protein Conformational Transition in Hydrophobic Charge Induction Chromatography: A Molecular Dynamics Simulation

Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead beta-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC.

Synthesis of Ordered Conducting Polymer Ultrathin Film and Its Application as Hole Injection Layer for Organic Light-Emitting Diode

A novel layer-ordered ultrathin film of poly(3,4-ethylene dioxythiophene)/arachidic acid (PEDOT:AA) was prepared by a modified Langmuir-Blodgett (LB) technique and an ordered conducting polymer embedded multilayer structure was constructed. The layer-ordered structure was deposited on an indium-tin oxide (ITO) surface as a hole injection layer for use as an organic light-emitting diode (OLED) and the luminance performance of the ITO/(PEDOT: PSS)/MEH-PPV(poly(2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene-vinylene))/Al device was investigated. Compared with electrostatic self-assembly (ESA) and spin-coating the poly(3,4-ethylene dioxythiophene)/polystyrenesulfonate (PEDOT:PSS) film showed enhanced luminance efficiency. The higher luminance at an equivalent voltage and lower turn-on voltage was observed after the PEDOT:AA film was utilized as hole injection layer in OLED. The enhanced carrie injection efficiency was attributed to the good layer-ordered structure of PEDOT:AA film, which decreased the contact resistance between ITO and the emitting layer. Further investigation revealed that this layer-ordered conducting polymer film exhibited poor temperature stability because of weak hydrophilic and hydrophobic interactions between adjacent LB layers. These weak interactions result in the film structure changing from a layer-ordered structure into an unordered structure at higher temperatures.

Head-to-tail Aggregates of Sulfonatomethylated Calix[4]resorcinarene in Aqueous Solutions

Two amphiphilic water-soluble sulfonatomethylated calix[4]resorcinarene derivatives were studied by various 1H NMR techniques (1H NMR titration, 2D NOESY, NMR diffusion measurements). The derivative with methyl moieties at the lower rim (1) was found to be non-aggregated in the range 0–10 mM in aqueous solutions. Lengthening of the lower rim substituent to pentyl (2) results in self-aggregation of 2 in aqueous solutions with the aggregation number varying from 3 at 1 mM to 20 at 10 mM. The 2D NOESY 1H NMR spectroscopy data reveal an unusual head-to-tail packing mode in aqueous solutions, resulting from the cooperative effect of weak hydrophobic interactions. Binding of guests (tetramethylammonium and N-methylpyridinium) results in additional stabilization of the aggregates whilst the head-to-tail packing mode of the aggregate is retained.

Structures and Free-Energy Landscapes of the Wild Type and Mutants of the Aβ 21–30 Peptide Are Determined by an Interplay between Intrapeptide Electrostatic and Hydrophobic Interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ 21–30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated p K a values that compare well with the experimental estimates. The p K a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22–K28 are more probable in “dried” salt bridges, whereas D23–K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ 21–30 peptide into two basins, differentiated by the relative position of the DVG(23–25) and GSN(25–27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.

Novel separation of bioactive catechin derivatives from complex plant mixtures by anion-exchange chromatography

The paper presents the use of anion-exchange chromatography to separate bioactive cysteine containing catechin conjugates from underivatised polyphenols and oligomeric material. The conjugates were excluded from the anion-exchange resin using a low percentage of organic solvent, enough to eliminate the weak hydrophobic interactions with the matrix. The rest of the uncharged polyphenols were unexpectedly retained by the matrix and eluted by the addition of salt. Cation–π interactions between the quaternary amino group on the resin and the electron-rich faces of the flavanol's aromatic rings might be responsible for this unexpected behaviour. Because immobilized anions retain uncharged polyphenolic material, anion-exchange chromatography may be a convenient approach to separate polyphenols from other bioactive compounds in complex mixtures of plant origin.

Synthesis of high selectivity polymeric adsorbent and its application on the separation of ginkgo flavonol glycosides and terpene lactones

A kind of weak hydrophobic adsorbent with functional group was synthesized. The relationship between the structure of the adsorbents and their adsorption properties was investigated in detail. With decrease of the content of non-polarity moiety in the adsorbent, i.e. divinylbenzene (DVB), the hydrophobic affinity of the adsorbent decreased. And then the adsorbent with appropriate DVB moiety content and functional group, i.e. amide group, can selectively adsorb flavonol glycosides based mainly on the synergistic mechanism of hydrogen bonding and weak hydrophobic interaction, while showing a fairly low adsorption capacity for terpene lactones by very weak hydrophobic affinity alone. In this study it was found that the adsorbent with amide group containing 8% DVB moiety would be favorable to separate the flavonol glycosides and terpene lactones in the extract of ginkgo leaves. After the adsorption and desorption process, the ratio of the weight of flavonal glycosides and terpene lactones in desorption solution was improved from 3.94:1 to 99.3:1, and the ratio of the weight of flavonal glycosides and terpene lactones in effluent solution was reduced from 3.94:1 to 0.114:1. The FTIR spectrum also verified the existence of the hydrogen bonding interaction between the adsorbent and the flavonol glycosides, just as expected. The adsorption mechanism was indicated to be the synergistic effect of the hydrogen bonding and the hydrophobic interaction.

Effects of mutations in the β subunit hinge domain on ATP synthase F 1 sector rotation: Interaction between Ser 174 and Ile 163

A complex of γ, ε, and c subunits rotates in ATP synthase (F oF 1) coupling with proton transport. Replacement of βSer174 by Phe in β-sheet4 of the β subunit (βS174F) caused slow γ subunit revolution of the F 1 sector, consistent with the decreased ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F1 sector. Stochastic fluctuation and a key domain of the β subunit, J. Biol. Chem. 282 (2007) 20698–20704]. Modeling of the domain including β-sheet4 and α-helixB predicted that the mutant βPhe174 residue undergoes strong and weak hydrophobic interactions with βIle163 and βIle166, respectively. Supporting this prediction, the replacement of βIle163 in α-helixB by Ala partially suppressed the βS174F mutation: in the double mutant, the revolution speed and ATPase activity recovered to about half of the levels in the wild-type. Replacement of βIle166 by Ala lowered the revolution speed and ATPase activity to the same levels as in βS174F. Consistent with the weak hydrophobic interaction, βIle166 to Ala mutation did not suppress βS174F. Importance of the hinge domain [phosphate-binding loop (P-loop)/α-helixB/loop/β-sheet4, βPhe148–βGly186] as to driving rotational catalysis is discussed.

Initiation and Propagation of Spectrin Heterodimer Assembly Involves Distinct Energetic Processes

Red cell spectrin alpha and beta subunits consist primarily of many tandem homologous motifs with very similar three- helix-bundle structures and similar dimer interfaces. Although misassembled homodimers can form under some conditions, correctly aligned heterodimers consistently assemble provided a small "dimer initiation" site near the actin binding domain is present. The dimer initiation site has been characterized to some extent, but little is known about the subsequent, low-affinity lateral interactions of the remaining motifs along the length of this ropelike molecule or the forces involved in these two steps of the dimerization process. In this study, we used isothermal titration calorimetry to deduce the mechanism and energetics of the two heterodimer assembly phases. The high-affinity initiation of dimerization is primarily enthalpically driven, which is consistent with initial alignment and docking of specific complementary alpha and beta motifs in the dimer initiation site driven by long-range electrostatic interactions followed by tight binding stabilized by hydrogen bonds and other hydrophilic interactions. In contrast, the subsequent weak lateral associations of additional motifs are primarily entropically driven, suggesting binding primarily involves weak hydrophobic interactions. Although initial docking is largely electrostatic, the only lateral interaction within the first four pairs of motifs that involves a net change in protons is the interaction of the alpha18 and beta4 repeats. This substoichiometric uptake of protons could be due to a pKa shift of a histidine in the alpha18 motif located near the dimer interface in a proposed homology-based model. On the basis of this analysis of heterodimer thermodynamics, a detailed model of spectrin dimer assembly is proposed.

Interfacial Property Modulation of Thermoresponsive Polymer Brush Surfaces and Their Interaction with Biomolecules

Dense poly(N-isopropylacrylamide) (PIPAAm) brushes were created on silica bead surfaces by surface-initiated atom transfer radical polymerization (ATRP). Interfacial properties of PIPAAm brushes were characterized by thermoresponisve interaction with biomolecules. The grafted amounts of PIPAAm on silica bead surfaces exceeded that from previously reported polymer-hydrogel-modified silica beads prepared by conventional radical polymerization by nearly 1 order of magnitude. Temperature-dependent chromatographic interactions with soluble analytes were modulated by changing the grafted PIPAAm chain lengths. Short PIPAAm-grafted silica beads produce insufficient dehydration and chain aggregation to separate steroids using weak hydrophobic interactions. In contrast, broad unresolved peaks were observed on silica beads column grafted with long PIPAAm chains due to steroid partitioning into thick, densely grafted PIPAAm brush layers. Thus, silica beads column grafted with PIPAAm chains of proper length can demonstrate baseline separation of steroids with relatively high resolution among the tested columns. Relatively longer retention times for steroid analytes were observed on all columns compared to those previously reported for other PIPAAm-grafted silica beads. This indicates that densely PIPAAm-grafted chains enable control of strong hydrophobic interactions with steroids by changing the column temperature. Densely grafted PIPAAm columns were also successful in separating two peptides into two peaks as the column temperature was increased to 40 degrees C. This provides an effective separation alternative for peptides using substantial hydrophobicity without modification of hydrophobic surfaces and/or low mobile phase pH. In conclusion, densely PIPAAm-grafted surfaces exhibit strong, reversible temperature-modulated hydrophobic interactions, facilitating baseline separations of steroids and peptides in aqueous milieu without changes in the mobile phase pH and high ionic strength.

Separation of basic and acidic compounds by capillary electrochromatography using monolithic silica capillary columns with zwitterionic stationary phase

A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.

On the microstructures of ethyl acetate/monoolein/water

A phase diagram, describing the behavior of the polar lipid monoolein (MO), water, and ethyl acetate (EtAc), is here presented as well as results from small angle X-ray scattering. MO is found to have a solubility of 60 wt.% in EtAc at 20 °C. No macroscopic aggregation of MO can, initially, be detected in the binary MO/EtAc solution even though MO forms solid crystals in concentrated samples when times goes by. In case of the ternary system small amounts of water, mainly bound to the lipid head groups, can be incorporated in the liquid EtAc/MO phase as water has a limited solubility in EtAc. For EtAc/water mass ratios below 2/3 EtAc is present into the reversed bicontinuous cubic and lamellar phases present in the binary MO/water system. To conclude, EtAc is mainly partitioned to the lipid membranes, with minor effects on spontaneous curvature. Hence, simple EtAc-addition has an effect similar to dehydration. For EtAc/water ratios above 2/3 the liquid crystalline phases dissolve. The phase behavior is here discussed in view of related phase behaviors for water-miscible solvent/MO/water systems. For instance, an interpretation of the swelling behavior of the sponge phase (L 3), present in the water-miscible solvent(s)/MO/water systems, shows that solvents partitioned to the polar domains strongly increases the spontaneous curvature of the MO-films. The reason is probably weaker hydrophobic interactions in interfacial regions. As expected, in case of water-miscible solvents, the ternary phase behaviors can be understood by consider water and water-miscible solvent as one “mixed solvent”.

DPM1, the Catalytic Subunit of Dolichol-phosphate Mannose Synthase, Is Tethered to and Stabilized on the Endoplasmic Reticulum Membrane by DPM3

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.