Micelle-bound State Research Articles

Expulsion of a potent cancer-cell photosensitizer from its micelle-bound state using β-cyclodextrin: A tenable model for efficient drug release.

The present study delves into the interaction of a potent cancer-cell photosensitizer Norharmane (NHM) with non-ionic triblock copolymer P123, followed by the assessment of the stability of the formed complex in the presence of β-cyclodextrin (β-CD). Spectroscopic results unveil the modulation of the prototropic equilibrium of NHM within the constrained microheterogeneous medium of the copolymer micelle to be favoured towards the neutral species of NHM over the cationic counterpart; which has been aptly rationalized invoking the key role of hydrophobic interaction in the association process and is further reinforced from steady-state and time-resolved spectroscopic measurements. The micropolarity of the probe-binding site has been evaluated by the archetypal ET(30) analysis revealing that the cationic probe remains in the corona region of the micelle instead of penetrating deeper into the micellar core. Moreover, the effect of β-CD on the stability of the NHM-bound P123 aggregates has also been investigated, revealing that β-CD can be used as a potential host for the release of the micelle-encapsulated drug through an inclusion complex formation with the P123 monomers. The result is expected to be of potential interest from medical perspective owing to the context of efficient drug release at their potential sites.

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR.

Lipid-based micellar nanoparticles promote aggregation of huntingtin exon-1 peptides. Here we characterize the interaction of two such peptides, httNTQ 7 and httNTQ 10 comprising the N-terminal amphiphilic domain of huntingtin followed by 7 and 10 glutamine repeats, respectively, with 8 nm lipid micelles using NMR chemical exchange saturation transfer (CEST), circular dichroism and pulsed Q-band EPR. Exchange between free and micelle-bound httNTQ n peptides occurs on the millisecond time scale with a KD ∼ 0.5-1 mM. Upon binding micelles, residues 1-15 adopt a helical conformation. Oxidation of Met7 to a sulfoxide reduces the binding affinity for micelles ∼3-4-fold and increases the length of the helix by a further two residues. A structure of the bound monomer unit is calculated from the backbone chemical shifts of the micelle-bound state obtained from CEST. Pulsed Q-band EPR shows that a monomer-dimer equilibrium exists on the surface of the micelles and that the two helices of the dimer adopt a parallel orientation, thereby bringing two disordered polyQ tails into close proximity which may promote aggregation upon dissociation from the micelle surface.

Backbone and Cbeta assignments for tau K18 in free and SDS micelle-bound states

Backbone and Cbeta assignments for tau K18 in free and SDS micelle-bound states

Structural transitions in tau k18 on micelle binding suggest a hierarchy in the efficacy of individual microtubule‐binding repeats in filament nucleation

The protein tau is found in an aggregated filamentous state in the intraneuronal paired helical filament deposits characteristic of Alzheimer's disease and other related dementias and mutations in tau protein and mRNA cause frontotemproal dementia. Tau isoforms include a microtubule-binding domain containing either three or four imperfect tandem microtubule binding repeats that also form the core of tau filaments and contain hexapaptide motifs that are critical for tau aggregation. The tau microtubule-binding domain can also engage in direct interactions with detergents, fatty acids, or membranes, which can greatly facilitate tau aggregation and may also mediate some tau functions. Here, we show that the alternatively spliced second microtubule-binding repeat exhibits significantly different structural characteristics compared with the other three repeats in the context of the intact repeat domain. Most notably, the PHF6* hexapeptide motif located at the N-terminus of repeat 2 has a lower propensity to form strand-like structure than the corresponding PHF6 motif in repeat 3, and unlike PHF6 converts to partially helical structure in the micelle-bound state. Interestingly, the behavior of the Module-B motif, located at the beginning of repeat 4, resembles that of PHF6* rather than PHF6. Our observations, combined with previous results showing that PHF6* and Module-B are both less effective than PHF6 in nucleating tau aggregation, suggest a hierarchy in the efficacy of these motifs in nucleating tau aggregation that originates in differences in their intrinsic propensities for extended strand-like structure and the resistance of these propensities to changes in tau's environment.

Micelle-bound structure of an extracellular Met-rich domain of hCtr1 and its binding with silver

Silver is a toxic metal. It can accumulate in tissues as a contaminant in foods and air. The human copper transporter 1 (hCtr1), as an uptake protein of copper, plays an important role in the accumulation of silver in mammalian cells. The extracellular N-terminal domain of hCtr1 contains two methionine-rich motifs, in which the second methionine-rich motif (M2) has been demonstrated to be crucial for uptake of Ag(I) and Cu(I). In the present work, the structure of an M2-inclusive peptide from hCtr1 34–53 in SDS (sodium dodecyl sulfate) micellar solution and the interaction of the micelle-bound peptide with Ag(I) were studied using nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC) and circular dichroism (CD) methods. The details of the binding site, affinity, stoichiometry and thermodynamics of the interaction between the micelle-bound peptide and Ag(I) were explored using NMR and calorimetric measurements titrated by Ag(I), and the role of each methionine residue in the binding of M2 motif with Ag(I) was estimated by individual substitution of methionine by alanine. It was revealed that the hydrophobic C-terminal region of the peptide folds as an α-helix and is embedded in SDS micelles, while the N-terminal region containing the Met motif locates at the surface of the micelles and binds Ag(I) mostly by Met40, Met41, Met43 and Met45. Differences between the peptide in the micelle-bound state and the peptide in the solution state with regards to the binding affinity, stoichiometry and thermodynamics were observed. It is suggested that the interaction between the extracellular domain of hCtr1 and membrane surface may play an important role for the binding structure and binding affinity of the M2 motif with Ag(I).

Binding of solvent molecules by micelles of ionic surfactants

The distribution of molecules between the free (intermicellar) and the micelle-bound states is found from the results of selective measurements of their self-diffusion coefficients in micellar solutions of ionic surfactants, i.e., sodium dodecylsulfate and cetyltrimethylammonium bromide.

Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe6, Tyr10, Leu13, and Met17 found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water–micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.

NMR determination of pKa values in α‐synuclein

The intrinsically unfolded protein α-synuclein has an N-terminal domain with seven imperfect KTKEGV sequence repeats and a C-terminal domain with a large proportion of acidic residues. We characterized pK(a) values for all 26 sites in the protein that ionize below pH 7 using 2D (1) H-(15) N HSQC and 3D C(CO)NH NMR experiments. The N-terminal domain shows systematically lowered pK(a) values, suggesting weak electrostatic interactions between acidic and basic residues in the KTKEGV repeats. By contrast, the C-terminal domain shows elevated pK(a) values due to electrostatic repulsion between like charges. The effects are smaller but persist at physiological salt concentrations. For α-synuclein in the membrane-like environment of sodium dodecylsulfate (SDS) micelles, we characterized the pK(a) of His50, a residue of particular interest since it is flanked within one turn of the α-helix structure by the Parkinson's disease-linked mutants E46K and A53T. The pK(a) of His50 is raised by 1.4 pH units in the micelle-bound state. Titrations of His50 in the micelle-bound states of the E46K and A53T mutants show that the pK(a) shift is primarily due to interactions between the histidine and the sulfate groups of SDS, with electrostatic interactions between His50 and Glu46 playing a much smaller role. Our results indicate that the pK(a) values of uncomplexed α-synuclein differ significantly from random coil model peptides even though the protein is intrinsically unfolded. Due to the long-range nature of electrostatic interactions, charged residues in the α-synuclein sequence may help nucleate the folding of the protein into an α-helical structure and confer protection from misfolding.

Structural Basis for the Association of the Redox-sensitive Target of Rapamycin FATC Domain with Membrane-mimetic Micelles

The target of rapamycin (TOR) is a conserved eukaryotic Ser/Thr kinase that regulates cellular growth in response to the nutrient and energy state. TOR signaling plays an important role in the development of diseases such as cancer, obesity, and diabetes and in different redox-sensitive processes (hypoxia, apoptosis, and aging). Because TOR has been detected at different cellular membranes and in the nucleus, its localization may influence the specific signaling readout. To better understand how TOR can associate with different membranes, the lipid-binding properties of the redox-sensitive yeast TOR1 FATC domain (y1fatc) have been characterized by solution NMR spectroscopy. Binding studies with different lipids indicate that y1fatc interacts specifically with a membrane-mimetic environment but appears not to recognize a specific lipid headgroup. In both, the structures of oxidized and reduced micelle-bound y1fatc, residues Ile-2456 to Trp-2470 of the lipid-binding motif form a hydrophobic bulb that has a rim of charged residues. The diffusion constants for both micelle-bound states are consistent with the rotational correlation times from the analysis of the (15)N relaxation data. Based on the K(d) values, the oxidized form (K(d) approximately 0.31 mm) binds dodecyl phosphocholine micelles slightly tighter than the reduced form (K(d) approximately 1.86 mM). Binding studies with y1fatc in which one or both tryptophans (Trp-2466 and Trp-2470) were replaced by alanine suggest that these residues are important for the exact positioning in the membrane and that the other aromatic (His-2462, Tyr-2463, and Phe-2469) and aliphatic residues (Ile-2456, Leu-2459, Ile-2464, and Pro-2468) in the lipid-binding motif contribute significantly to the affinity.

New Insights into the Interfacial Activation of Secreted Phospholipase A2

Despite numerous studies towards elucidation of the structural basis of activation of secreted PLA2s upon membrane binding (interfacial activation), no consistent or clear picture has emerged thus far. Previously we have reported significant changes in the secondary and dynamic structures of human group IB and IIA PLA2s, as well as changes in their mode of membrane binding during activation. Here we have conducted atomic resolution NMR studies on free and phospholipid micelle-bound human group IIA PLA2 (hIIAPLA2) to detect more detailed molecular events underlying interfacial activation. Two-dimensional 1H,15N-HSQC spectra have been obtained at 600 MHz on Ca2+-free and Ca2+-loaded hIIAPLA2 in the presence of dodecylphosphocholine (DPC) micelles. Upon complex formation with the micelles, signals from arginine side chain -NH2 groups of Ca2+-loaded hIIAPLA2 are observed, whereas for Ca2+-free PLA2 these signals are absent because of fast H/D exchange with the solvent. This suggests that the Ca2+-loaded hIIAPLA2 tightly binds to the micelles so these groups are sequestered at the PLA2-micellar interface and shielded from the solvent, or that they are otherwise stabilized by strong hydrogen bonding in the micelle-bound state. TROSY experiments (900 MHz) on Ca2+-loaded, 15N,13C-labeled hIIAPLA2 in the absence and presence of DPC micelles (1:600 protein-to-DPC molar ratio) identify substantial conformational changes in PLA2 upon binding to the micelles. Based on the assigned chemical shifts, important structural changes occur throughout the protein. The molecular mechanism of the strong increase in activity of hIIAPLA2 upon phospholipid surface binding is likely to involve a widening of the substrate binding pocket, mediated by a rigid-body movement of the N-terminal helix via interactions of the cationic residues (e.g., Arg7) with lipid phosphate groups. This mechanism will be tested in further studies and may be shared by other secreted PLA2 isoforms.

VDAC Studied by Solution NMR: Implications for the Native Structure

The voltage-dependent anion channel (VDAC) is the main pathway for metabolites, small molecules and ions across the eukaryotic outer mitochondrial membrane. VDAC has been extensively studied for over thirty years and recently, high-resolution structures of VDAC were determined by X-ray and NMR methods (1-3). These studies used recombinant, refolded protein in membrane mimicking environments and thus the valid question arises how well the resulting atomic structure might resemble the “native” structure of VDAC in the mitochondrial outer membrane (4).Here, we describe implications from solution NMR experiments to this question. Recombinant human VDAC-1 is stably folded in LDAO detergent micelles. Well resolved NMR spectra, including four-dimensional NOESYs, yielded a consistent set of more than 1000 spatial spin-spin correlations that unambiguously define the three-dimensional structure of VDAC-1 (1). The protein forms a 19-stranded beta-barrel with 18 antiparallel and 1 parallel strand pairing. The N-terminal 25 residues are not part of the beta-barrel and solution NMR data link the dynamic properties of this segment to the well-known voltage gating process. The inner diameter of the VDAC-1 barrel is about 25 A, in consistence with published micrographs of native or native-like preparations. The entire outside perimeter of the barrel is hydrophobic and covered by detergent molecules, compatible with a membrane bilayer topology. NMR measurements also revealed interactions of VDAC-1 with beta-NADH and cholesterol, providing a functional connection to experiments on native states of the protein. Furthermore, we can link the micelle-bound state of VDAC structurally and functionally to preparations in phospholipid bilayers by comparing NMR spectra and electron micrographs.(1) Hiller et al. Science 321, 1206 (2008).(2) Bayrhuber et al. PNAS 105, 15370 (2008).(3) Ujwal et al. PNAS 105, 17742 (2008).(4) Colombini. Trends Biochem. Sci. 34, 382 (2009).

Effect of Pseudorepeat Rearrangement on α-Synuclein Misfolding, Vesicle Binding, and Micelle Binding

The pathological and physiological hallmarks of the protein α-synuclein (aS) are its misfolding into cytotoxic aggregates and its binding to synaptic vesicles, respectively. Both events are mediated by seven 11-residue amphiphilic pseudorepeats and, most generally, involve a transition from intrinsically unstructured conformations to structured conformations. Based on aS interactions with aggregation-inhibiting small molecules, an aS variant termed shuffled α-synuclein (SaS), wherein the first six pseudorepeats had been rearranged, was introduced. Here, the effects of this rearrangement on misfolding, vesicle binding, and micelle binding are examined in reference to aS and β-synuclein to study the sequence characteristics underlying these processes. Fibrillization correlates with the distinct clustering of residues with high β-sheet propensities, while vesicle affinities depend on the mode of pseudorepeat interchange and loss. In the presence of micelles, the pseudorepeat region of SaS adopts an essentially continuous helix, whereas aS and β-synuclein encounter a distinct helix break, indicating that a more homogeneous distribution of surfactant affinities in SaS prevented the formation of an extensive helix break in the micelle-bound state. By demonstrating the importance of the distribution of β-sheet propensities and by revealing inhomogeneous aS surfactant affinities, the present study provides novel insights into two central themes of synuclein biology.

The Importance of Micelle-Bound States for the Bioactivities of Bifunctional Peptide Derivatives for δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists

To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of delta-preferring opioid agonists and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2; 2: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF 3) 2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications.

1H, 13C, and 15N resonance assignment of the cAMP-regulated phosphoprotein endosulfine-alpha in free and micelle-bound states

(13)C, (15)N, and (1)H chemical shift assignments are presented for the cAMP-regulated phosphoprotein endosulfine-alpha in its free and micelle-bound states. Secondary chemical shift analysis demonstrates formation of four helices in the micelle-bound state, which are not present in the absence of detergent.

Comparison of Structure and Dynamics of Micelle-bound Human α-Synuclein and Parkinson Disease Variants

Three point mutations (A30P, E46K, and A53T) as well as gene triplication genetically link the 140-residue protein alpha-synuclein (aS) to the development of Parkinson disease. Here, the structure and dynamics of micelle-bound aS(A30P) and aS(A53T) are described and compared with wild-type aS, in addition to describing the aS-micelle interaction. A53T is sensed only by directly adjacent residues and leaves the backbone structure and dynamics indistinguishable from the wild type. A30P interrupts one helix turn (Val26-Ala29) and destabilizes the preceding one. A shift in helix register following A30P disturbs the canonical succession of polar and hydrophobic residues for at least two turns. The shortened helix-N adopts a slightly higher helical content and is less bent, indicating that strain was present in the micelle-bound helix. In the vicinity of the A30P-induced perturbations, the underlying micelle environment has rearranged, but nevertheless all aS variants maintain similar interrelationships with the micelle. Moreover, aS-micelle immersion correlates well with fast and slow aS backbone dynamics, allowing a rare insight into protein-micelle interplay.

Structure and Dynamics of Micelle-bound Human α-Synuclein

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

Mycobacterium tuberculosis chaperonin 10 is secreted in the macrophage phagosome: is secretion due to dissociation and adoption of a partially helical structure at the membrane?

To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a beta-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately beta-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment.

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Membrane binding motif of the P-type cardiotoxin

Carditoxins (CTXs) from cobra snake venoms, the basic 60–62 residue all-beta sheet polypeptides, are known to bind to and impair the function of cell membranes. To assess the membrane induced conformation and orientation of CTXs, the interaction of the P-type cardiotoxin II from Naja oxiana snake venom (CTII) with perdeuterated dodecylphosphocholine (DPC) was studied using 1H-NMR spectroscopy and diffusion measurements. Under conditions where the toxin formed a well-defined complex with DPC, the spatial structure of CTII with respect to the presence of tightly bound water molecules in loop II, was calculated using the torsion angle dynamics program DYANA. The structure was found to be similar, except for subtle changes in the tips of all three loops, to the previously described “major” form of CTII in aqueous solution illustrated by the “trans” configuration of the Val7-Pro8 peptide bond. No “minor” form with the “ cis” configuration of the above bond was found in the micelle-bound state. The broadening of the CTII backbone proton signals by 5, 16-doxylstearate relaxation probes, together with modeling based on the spatial structure of CTII, indicated a periphery mode of binding of the toxin molecule to the micelle and revealed its micelle interacting domain. The latter includes a hydrophobic region of CTII within the extremities of loops I and III (residues 5–11, 46–50), the basement of loop II (residues 24–29,31–37) and the belt of polar residues encircling these loops (lysines 4,5,12,23,50, serines 11,46, histidine 31, arginine 36). It is suggested that this structural motif and the mode of binding can be realized during interaction of CTXs with lipid and biological membranes.

Unfolding and refolding of cytochrome c driven by the interaction with lipid micelles.

Binding of native cyt c to L-PG micelles leads to a partially unfolded conformation of cyt c. This micelle-bound state has no stable tertiary structure, but remains as alpha-helical as native cyt c in solution. In contrast, binding of the acid-unfolded cyt c to L-PG micelles induces folding of the polypeptide, resulting in a similar helical state to that originated from the binding of native cyt c to L-PG micelles. Far-ultraviolet (UV) circular dichroism (CD) spectra showed that this common micelle-associated helical state (HL) has a native-like alpha-helix content, but is highly expanded without a tightly packed hydrophobic core, as revealed by tryptophan fluorescence, near-UV, and Soret CD spectroscopy. The kinetics of the interaction of native and acid-unfolded cyt c was investigated by stopped-flow tryptophan fluorescence. Formation of H(L) from the native state requires the disruption of the tightly packed hydrophobic core in the native protein. This micelle-induced unfolding of cyt c occurs at a rate approximately 0.1 s(-1), which is remarkably faster in the lipid environment compared with the expected rate of unfolding in solution. Refolding of acid-unfolded cyt c with L-PG micelles involves an early highly helical collapsed state formed during the burst phase (<3 ms), and the observed main kinetic event reports on the opening of this early compact intermediate prior to insertion into the lipid micelle.