Novel intracellular delivery system of antisense oligonucleotide by self-assembled hybrid micelles composed of DNA/PEG conjugate and cationic fusogenic peptide

An antisense oligonucleotide (ODN), c-myb, was covalently conjugated to poly(ethylene glycol) (PEG) via an acid-cleavable phosphoramidate linkage to form a diblock copolymer-like structure. The phosphoramidate linkage between ODN and PEG was completely cleaved within 5 h in an endosomal acidic condition (pH 4.7). When complexed with a cationic fusogenic peptide, KALA, the ODN/PEG conjugate self-associated to form polyelectrolyte complex micelles in an aqueous solution. The anionic ODN segments were ionically interacted with cationic KALA peptide to form an inner polyelectrolyte complex core, while the PEG segments constituted a surrounding corona. Effective hydrodynamic volume of the micelles was ca. 70 nm with a very narrow size distribution. The polyelectrolyte complex micelles, composed of c-myb ODN-PEG conjugate and KALA, were transported into cells far more efficiently than c-myb ODN itself. They also exhibited higher antiproliferative activity against smooth muscle cells. This study demonstrates that the DNA/PEG hybrid micelles system can be applied for the delivery of antisense oligonucleotide.     

Self-assembly of polypeptide-containing ABC-type triblock copolymers in aqueous solution and its pH dependence

Self-assembling of novel biodegradable ABC-type triblock copolymer poly(ethylene glycol)-poly(L-lactide)-poly(L-glutamic acid) (PEG-PLLA-PLGA) is studied. In aqueous media, it self-assembles into a spherical micelle with the hydrophobic PLLA segment in the core and the two hydrophilic segments PEG and PLGA in the shell. With the lengths of PEG and PLLA blocks fixed, the diameter of the micelles depends on the length of the PLGA block and on the volume ratio of H(2)O/dimethylformamide (DMF) in the media. When the PLGA block is long enough, morphology of the self-assembly is pH-dependent. It assembles into the spherical micelle in aqueous media at pH 4.5 and into the connected rod at or below pH 3.2. The critical micelle concentration (cmc) of the copolymer changes accordingly with decreasing solution pH. Both aggregation states can convert to each other at the proper pH value. This reversibility is ascribed to the dissociation and neutralization of the COOH groups in the LGA residues. When the PLGA block is short compared to the PEG or PLLA block, it assembles only into the spherical micelle at various pH values.     

pH-responsive oligodeoxynucleotide (ODN)-poly(ethylene glycol) conjugate through acid-labile beta-thiopropionate linkage: preparation and polyion complex micelle formation

An oligodeoxynucleotide (ODN) conjugated to poly(ethylene glycol) (PEG) through a pH-responsive ester linkage (PEG-ODN conjugate) was successfully synthesized by the Michael reaction of 3'-thiol-modified ODN with a heterobifunctional PEG bearing an acetal group at the alpha-end and an acrylate group at the omega-end (acetal-PEG-acrylate), aimed at the development of a novel ODN delivery system. The prepared PEG-ODN conjugate and linear-poy(ethyleneimine) (L-PEI) spontaneously associated to form a polyion complex (PIC) micelle whose diameter and polydispersity index micro(2)/Gamma(2)) were 102.5 nm and 0.096 as determined by DLS measurements, respectively. Both the PEG-ODN conjugate and PIC micelle showed cleavage of the ester linkage at the endosomal pH (=5.5), suggesting that the PIC micelle is anticipated to release the ODN in the intracellular compartment. Furthermore, the PEG-ODN conjugate in the PIC micelle was stable against deoxyribonuclase (DNase I) digestion and has no interaction with the serum component because of the steric stabilization of the highly dense PEG corona surrounding the PIC core. These characteristics of the PIC micelles entrapping the PEG-ODN conjugate are promising for their utility as a novel ODN delivery system.     

Bioreducible micelles self-assembled from amphiphilic hyperbranched multiarm copolymer for glutathione-mediated intracellular drug delivery

A new type of biodegradable micelles for glutathione-mediated intracellular drug delivery was developed on the basis of an amphiphilic hyperbranched multiarm copolymer (H40-star-PLA-SS-PEP) with disulfide linkages between the hydrophobic polyester core and hydrophilic polyphosphate arms. The resulting copolymers were characterized by nuclear magnetic resonance (NMR), Fourier transformed infrared (FTIR), gel permeation chromatography (GPC), and differential scanning calorimeter (DSC) techniques. Benefiting from amphiphilic structure, H40-star-PLA-SS-PEP was able to self-assemble into micelles in aqueous solution with an average diameter of 70 nm. Moreover, the hydrophilic polyphosphate shell of these micelles could be detached under reduction-stimulus by in vitro evaluation, which resulted in a rapid drug release due to the destruction of micelle structure. The glutathione-mediated intracellular drug delivery was investigated against a Hela human cervical carcinoma cell line. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements demonstrated that H40-star-PLA-SS-PEP micelles exhibited a faster drug release in glutathione monoester (GSH-OEt) pretreated Hela cells than that in the nonpretreated cells. Cytotoxicity assay of the doxorubicin-loaded (DOX-loaded) micelles indicated the higher cellular proliferation inhibition against 10 mM of GSH-OEt pretreated Hela cells than that of the nonpretreated ones. As expected, the DOX-loaded micelles showed lower inhibition against 0.1 mM of buthionine sulfoximine (BSO) pretreated Hela cells. These reduction-responsive and biodegradable micelles show a potential to improve the antitumor efficacy of hydrophobic chemotherapeutic drugs.     

Polyelectrolyte complex micelles composed of c-raf antisense oligodeoxynucleotide-poly(ethylene glycol) conjugate and poly(ethylenimine): effect of systemic administration on tumor growth

An antisense oligodeoxynucleotide (ODN) delivery system based on polyelectrolyte complex (PEC) micelles composed of an ODN-poly(ethylene glycol) (PEG) conjugate and polyethylenimine (PEI) was demonstrated. The PEC micelles having a core/shell structure were spontaneously formed in an aqueous solution by ionic interactions between ODN part in the conjugate and PEI. The ODN/PEI polyelectrolyte complex formed an inner core while PEG chains surrounded it as a shell. The morphology of the micelles was visualized as a separate sphere by atomic force microscopy (AFM). When the micelles containing a c-raf antisense ODN were intravenously administered into tumor-bearing nude mice, significant antitumor activities against human lung cancer were observed. The intravenously injected micelles also showed significantly higher accumulation level in the solid tumor region compared to that of naked ODN.     

A carbosilane dendrimer and a silacyclopentadiene analog carrying peripheral lactoses as drug-delivery systems

A carbosilane dendrimer (4a) and its silacyclopentadiene analog (4b), both functionalized with lactoses, were tested for their abilities to act as drug-delivery systems. The critical micelle concentrations of 4a and 4b were measured using the drop-volume method in water and were 1.7 and 2.9 μM, respectively, suggesting that they could act as aggregates of glycoclusters. The amounts of the hydrophobic dye Orange OT loaded onto aqueous micelles of 4a and 4b and the stabilities of the dye/micelle complexes were determined by extracting the dyes from the complexes into chloroform. The particle sizes were measured for the loaded micelles by dynamic light scattering. Transfer of the dye from the micelles to peanut agglutinin was observed by fluorescence microscopy. Given the abilities of micelles of 4a and 4b to bind and release Orange OT, these glycocluster micelles may find use as drug-delivery systems.     

NMR studies of the anti-apoptotic protein Bcl-xL in micelles

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.     

Bioreducible block copolymers based on poly(ethylene glycol) and poly(γ-benzyl L-glutamate) for intracellular delivery of camptothecin

Poly(ethylene glycol)-b-poly(γ-benzyl L-glutamate)s bearing the disulfide bond (PEG-SS-PBLGs), which is specifically cleavable in intracellular compartments, were prepared via a facile synthetic route as a potential carrier of camptothecin (CPT). Diblock copolymers with different lengths of PBLG were synthesized by ring-opening polymerization of benzyl glutamate N-carboxy anhydride in the presence of a PEG macroinitiator (PEG-SS-NH(2)). Owing to their amphiphilic nature, the copolymers formed spherical micelles in an aqueous condition, and their particle sizes (20-125 nm in diameter) were dependent on the block length of PBLG. Critical micelle concentrations of the copolymers were in the range 0.005-0.065 mg/mL, which decreased as the block length of PBLG increased. CPT, chosen as a model anticancer drug, was effectively encapsulated up to 12 wt % into the hydrophobic core of the micelles by the solvent casting method. It was demonstrated by the in vitro optical imaging technique that the fluorescence signal of doxorubicin, quenched in the PEG-SS-PBLG micelles, was highly recovered in the presence of glutathione (GSH), a tripeptide reducing disulfide bonds in the cytoplasm. The micelles released CPT completely within 20 h under 10 mM GSH, whereas only 40% of CPT was released from the micelles in the absence of GSH. From the in vitro cytotoxicity test, it was found that CPT-loaded PEG-SS-PBLG micelles showed higher toxicity to SCC7 cancer cells than CPT-loaded PEG-b-PBLG micelles without the disulfide bond. Microscopic observation demonstrated that the disulfide-containing micelle could effectively deliver the drug into nuclei of SCC7 cells. These results suggest that PEG-SS-PBLG diblock copolymer is a promising carrier for intracellular delivery of CPT.     

Formation of core-shell type biodegradable polymeric micelles from amphiphilic poly(aspartic acid)-block-polylactide diblock copolymer

Poly(aspartic acid)-block-polylactide diblock copolymers (PAsp-b-PLAs) having both hydrophilic and hydrophobic segments of various lengths were synthesized. These PAsp-b-PLA diblock copolymers formed polymeric micelles consisting of a hydrophobic PLA core and a hydrophilic, pH-sensitive PAsp shell in aqueous solution. The effects of the segment length of both the PLA and the PAsp portions and the pH of the solution on the shapes and sizes of the PAsp-b-PLA polymeric micelles were investigated. The results indicated a balance between the effects of electrostatic repulsion, hydrogen bonding in the PAsp shell layer, and hydrophobic interactions in the PLA core determine the sizes of the PAsp-b-PLA polymeric micelles. Moreover, the PAsp-b-PLA polymeric micelles did not possess any cytotoxic activity against L929 fibroblast cells. The obtained polymeric micelle should be useful for biodegradable biomedical materials such as drug delivery vehicle.     

Structure, dynamics, and insertion of a chloroplast targeting peptide in mixed micelles

Nuclear-encoded, chloroplast-destined proteins are synthesized with transit sequences that contain all information to get them inside the organelle. Different proteins are imported via a general protein import machinery, but their transit sequences do not share amino acid homology. It has been suggested that interactions between transit sequence and chloroplast envelope membrane lipids give rise to recognizable, structural motifs. In this study a detailed investigation of the structural, dynamical, and topological features of an isolated transit peptide associated with mixed micelles is described. The structure of the preferredoxin transit peptide in these micelles was studied by circular dichroism (CD) and multidimensional NMR techniques. CD experiments indicated that the peptide, which is unstructured in aqueous solution, obtained helical structure in the presence of the micelles. By NMR it is shown that the micelles introduced ill-defined helical structures in the transit peptide. Heteronuclear relaxation experiments showed that the whole peptide backbone is very flexible. The least dynamic segments are two N- and C-terminal helical regions flanking an unstructured proline-rich amino acid stretch. Finally, the insertion of the peptide backbone in the hydrophobic interior of the micelle was investigated by use of hydrophobic spin-labels. The combined data result in a model of the transit peptide structure, backbone dynamics, and insertion upon its interaction with mixed micelles.     

"Schizophrenic" micellization associated with coil-to-helix transitions based on polypeptide hybrid double hydrophilic rod-coil diblock copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.     

Synthesis, characterization, antitumor activity of pluronic mimicking copolymer micelles conjugated with doxorubicin via acid-cleavable linkage

Pluronic mimicking poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer having multiple hydroxyl groups in the PPO middle segment (core-functionalized Pluronic: CF-PLU) was synthesized for conjugation of doxorubicin (DOX). DOX was conjugated on the multiple hydroxyl groups of CF-PLU via an acid-labile hydrazone linkage (CF-PLU-DOX). In aqueous solution, CF-PLU-DOX copolymers self-assembled to form a core/shell-type micelle structure consisting of a hydrophobic DOX-conjugated PPO core and a hydrophilic PEO shell layer. The conjugated DOX from CF-PLU-DOX micelles was released out more rapidly at pH 5 than pH 7.4, indicating that the hydrazone linkage was cleaved under acidic condition. CF-PLU-DOX micelles exhibited greatly enhanced cytotoxicity for MCF-7 human breast cancer cells compared to naked DOX, while CF-PLU copolymer itself showed extremely low cytotoxicity. Flow cytometry analysis revealed that the extent of cellular uptake for CF-PLU-DOX micelles was greater than free DOX. Confocal image analysis also showed that CF-PLU-DOX micelles had a quite different intracellular distribution profile from free DOX. CF-PLU-DOX micelles were mainly distributed in the cytoplasm, endosomal/lysosomal vesicles, and nucleus, while free DOX was localized mainly within the nucleus, suggesting that CF-PLU-DOX micellar formulation might be advantageously used for overcoming the multidrug resistance (MDR) effect, which gradually develops in many tumor cells during repeated drug administration.     

Interaction of fatty acid sodium salts with sodium deoxycholate

Interaction of homologous fatty acids (C3-C18) with sodium deoxycholate was investigated. From NMR and ultrasonic results it was found that short chain homologues (up to C9) do not participate in the formation of mixed micelles with sodium deoxycholate. Fatty acid homologues with longer chains (starting with C9) form mixed micelles by "burying" hydrophobic chains in hydrophobic environment of a sodium deoxycholate micelle.     

Self-assembled micelles of biodegradable triblock copolymers based on poly(ethyl ethylene phosphate) and poly(-caprolactone) as drug carriers

A series of novel amphiphilic triblock copolymers of poly(ethyl ethylene phosphate) and poly(-caprolactone) (PEEP-PCL-PEEP) with various PEEP and PCL block lengths were synthesized and characterized. These triblock copolymers formed micelles composed of a hydrophobic core of poly(-caprolactone) (PCL) and a hydrophilic shell of poly(ethyl ethylene phosphate) (PEEP) in aqueous solution. The micelle morphology was spherical, determined by transmission electron microscopy. It was found that the size and critical micelle concentration values of the micelles depended on both hydrophobic PCL block length and PEEP hydrophilic block length. The in vitro degradation characteristics of the triblock copolymers were investigated in micellar form, showing that these copolymers were completely biodegradable under enzymatic catalysis of Pseudomonas lipase and phosphodiesterase I. These triblock copolymers were used for paclitaxel (PTX) encapsulation to demonstrate the potential in drug delivery. PTX was successfully loaded into the micelles, and the in vitro release profile was found to be correlative to the polymer composition. These biodegradable triblock copolymer micelles are potential as novel carriers for hydrophobic drug delivery.     

Layer-by-layer Assembly of Poly(lactic-co-glycolic acid)-b-poly(l-lysine) Copolymer Micelles

Biocompatible, biodegradable polyionic micelles were used as a building component for layer-by-layer (LbL) assembly that can produce drug-loaded nanolayers. To prepare the polycationic micelles, poly(lactic-co-glycolic acid)-b-poly(l-lysine) [PLGA-b-P(Lys)] copolymers were synthesized. In an aqueous phase, PLGA-b-P(Lys) copolymers were self-assembled to form spherical micelles with the inner core of poly(lactic-co-glycolic acid) (PLGA) and the cationic outer shell of P(Lys). The micelles were characterized by zeta potential, dynamic light scattering, and nuclear magnetic resonance. PLGA-b-P(Lys) micelles showed the positive zeta potential values in a broad range of pH (3–11), indicating the high stability of the polyionic micelles with the outer shell of positive charges. Cationic polymeric micelles were coated on the surface via electrostatic interactions with the oppositely charged polyelectrolyte, poly(sodium 4-styrenesulfonate). Formation of multiple micelle layers was monitored using quartz crystal microbalance in situ, and the surface topology of the layers was characterized by atomic force microscopy ex situ, as the number of micelle layer was increased. The multiple micelle layers were stable, and the thickness of micelle layer grew as the number of LbL coating increased. The approach described in this work can be used for the development of the biocompatible, biodegradable, drug-loaded bioactive nanocoatings.     

Polymer terminal group effects on properties of thermoresponsive polymeric micelles with controlled outer-shell chain lengths

Well-defined amphiphilic diblock copolymers comprising thermoresponsive polymer segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PID) and hydrophobic polymer segments, poly(benzyl methacrylate) (PBzMA), were synthesized by controlled living radical polymerization. Terminal derivatization of PID segments to either hydroxyl or phenyl groups was achieved through reactions of coupling agents with thiol groups exposed by cleavage of terminal dithiobenzoate groups. Diblock copolymers formed core-shell type polymeric micelles with thermoresponsive outer shells. Hydrodynamic micellar diameters ranged from 12 to 31 nm, controlled by varying PID chain lengths. Differences in PID terminal groups did not affect the critical micelle concentration or micellar diameters. However, these groups demonstrated a significant influence on the micellar thermoresponses. Hydroxylated PID/PBzMA micelles exhibited a phase transition of approximately 40 degrees C, independent of PID molecular weights. Even though molecular weights and compositions of PID chains were equivalent except for terminal groups, micelles having the outermost surface phenyl groups exhibited drastically lower phase transition temperature shifts, especially for micelles with low molecular weight PID chains.     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

Mesoscale Simulation and cryo-TEM of Nanoscale Drug Delivery Systems

This work sets out to study the effect of hydrophobic molecules on the morphology of aqueous solutions of amphiphilic block copolymer, which has potential drug delivery applications. The effect is studied both experimentally and by using simulations. Using cryogenic TEM observations, micelles can clearly be visualised and their core size measured. While pure polymer solutions form into spherical micelles with a narrow size distribution, addition of small amounts of hydrophobic drug molecules leads to distortions in shape, a wider size distribution, and larger average core diameter. Simulations are based on a mesoscale dynamic density functional method with Gaussian chain Hamiltonian and mean-field interactions, as implemented in the MesoDyn code. With parameters for the amphiphilic system established in earlier work, and mean-field interactions for the drug molecule derived from structure–property relationships, we obtain good agreement with the TEM observations for the effect of the hydrophobic molecules on the morphology. The simulations clearly show how increasing drug concentration leads to an increase in micelle size, a wider distribution and more elongated rather than spherical micelles.     

Global fold and backbone dynamics of the hepatitis C virus E2 glycoprotein transmembrane domain determined by NMR

E1 and E2 are two hepatitis C viral envelope glycoproteins that assemble into a heterodimer that is essential for membrane fusion and penetration into the target cell. Both extracellular and transmembrane (TM) glycoprotein domains contribute to this interaction, but study of TM–TM interactions has been limited because synthesis and structural characterization of these highly hydrophobic segments present significant challenges. In this NMR study, by successful expression and purification of the E2 transmembrane domain as a fusion construct we have determined the global fold and characterized backbone motions for this peptide incorporated in phospholipid micelles. Backbone resonance frequencies, relaxation rates and solvent exposure measurements concur in showing this domain to adopt a helical conformation, with two helical segments spanning residues 717–726 and 732–746 connected by an unstructured linker containing the charged residues D728 and R730 involved in E1 binding. Although this linker exhibits increased local motions on the ps timescale, the dominating contribution to its relaxation is the global tumbling motion with an estimated correlation time of 12.3 ns. The positioning of the helix–linker–helix architecture within the mixed micelle was established by paramagnetic NMR spectroscopy and phospholipid-peptide cross relaxation measurements. These indicate that while the helices traverse the hydrophobic interior of the micelle, the linker lies closer to the micelle perimeter to accommodate its charged residues. These results lay the groundwork for structure determination of the E1/E2 complex and a molecular understanding of glycoprotein heterodimerization.