In vivo characterization of the biodistribution profile of amphipol A8-35.

Amphipols (APols) are polymeric surfactants that keep membrane proteins (MPs) water-soluble in the absence of detergent, while stabilizing them. They can be used to deliver MPs and other hydrophobic molecules in vivo for therapeutic purposes, e.g., vaccination or targeted delivery of drugs. The biodistribution and elimination of the best characterized APol, a polyacrylate derivative called A8-35, have been examined in mice, using two fluorescent APols, grafted with either Alexa Fluor 647 or rhodamine. Three of the most common injection routes have been used, intravenous (IV), intraperitoneal (IP), and subcutaneous (SC). The biodistribution has been studied by in vivo fluorescence imaging and by determining the concentration of fluorophore in the main organs. Free rhodamine was used as a control. Upon IV injection, A8-35 distributes rapidly throughout the organism and is found in most organs but the brain and spleen, before being slowly eliminated (10-20 days). A similar pattern is observed after IP injection, following a brief latency period during which the polymer remains confined to the peritoneal cavity. Upon SC injection, A8-35 remains essentially confined to the point of injection, from which it is only slowly released. An interesting observation is that A8-35 tends to accumulate in fat pads, suggesting that it could be used to deliver anti-obesity drugs.

High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase.

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.

Nanoparticle surface-enhanced Raman scattering of bacteriorhodopsin stabilized by amphipol A8-35.

Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee-Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 10(2). SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein-stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Quantitative studies on the localization of the cholinergic receptor protein in the normal and denervated electroplaque from Electrophorus electricus.

Electroplaques dissected from the electric organ of Electrophorus electricus are labeled by tritiated alpha1-isotoxin from Naja nigricollis, a highly selective reagent of the cholinergic (nicotinic) receptor site. Preincubation of the cell with an excess of unlabeled alpha-toxin and with a covalent affinity reagent or labeling in the presence of 10(-4) M decamethonium reduces the binding of [3H]alpha-toxin by at least 75%. Absolute surface densities of alpha-toxin sites are estimated by high-resolution autoradiography on the basis of silver grain distribution and taking into account the complex geopmetry of the cell surface. Binding of [3H]alpha-toxin on the noninnervated face does not differ from background. Labeled sites are observed on the innervated membrane both between the synapses and under the nerve terminals but the density of sites is approx. 100 times higher at the level of the synapses than in between. Analysis of the distance of silver grains from the innervated membrane shows a symmetrical distribution centered on the postsynaptic plasma membrane under the nerve terminal. In extrasynaptic areas, the barycenter of the distribution lies approximately 0.5 micrometer inside the cell, indicating that alpha-toxin sites are present on the membrane of microinvaginations, or caveolae, abundant in the extrajunctional areas. An absolute density of 49,600 +/- 16,000 sites/micrometer2 of postsynaptic membrane is calculated; it is in the range of that found at the crest of the folds at the neuromuscular junction and expected from a close packing of receptor molecules. Electric organs were denervated for periods up to 142 days. Nerve transmission fails after 2 days, and within a week all the nerve terminals disappear and are subsequently replaced by Schwann cell processes, whereas the morphology of the electroplaque remains unaffected. The denervated electroplaque develops some of the electrophysiological changes found with denervated muscles (increases of membrane resting resistance, decrease of electrical excitability) but does not become hypersensitive to cholinergic agonists. Autoradiography of electroplaques dissected from denervated electric organs reveals, after labeling with [3H]alpha-toxin, patches of silver grains with a surface density close to that found in the normal electroplaque. The density of alpha-toxin binding sites in extrasynaptic areas remains close to that observed on innervated cells, confirming that denervation does not cause an increase in the number of cholinergic receptor sites. The patches have the same distribution, shape,and dimensions as in subneural areas of the normal electroplaque, and remnants of nerve terminal or Schwann cells are often found at the level of the patches. They most likely correspond to subsynaptic areas which persist with the same density of [3H]alpha-toxin sites up to 52 days after denervation. In the adult synapse, therefore, the receptor protein exhibits little if any tendency for lateral diffusion.

Thermodynamic characterization of the exchange of detergents and amphipols at the surfaces of integral membrane proteins.

The aggregation of integral membrane proteins (IMPs) in aqueous media is a significant concern for mechanistic investigations and pharmaceutical applications of this important class of proteins. Complexation of IMPs with amphiphiles, either detergents or short amphiphilic polymers known as amphipols (APols), renders IMPs water-soluble. It is common knowledge that IMP-detergent complexes are labile, while IMP-APol complexes are exceptionally stable and do not dissociate even under conditions of extreme dilution. To understand the thermodynamic origin of this difference in stability and to guide the design of new APols, we have studied by isothermal titration calorimetry (ITC) the heat exchanges during two reciprocal processes, the "trapping" of detergent-solubilized IMPs in APols and the "stripping" of IMP-APol complexes by detergents, using two IMPs (the transmembrane domain of porin OmpA from Escherichia coli and bacteriorhodopsin from Halobium salinarium), two APols [an anionic polymer derived from acrylic acid (A8-35) and a cationic phosphorylcholine-based polymer (C22-43)], and two neutral detergents [n-octyl thioglucoside (OTG) and n-octyltetraethylene glycol (C(8)E(4))]. In the presence of detergent, free APols and IMP-APol complexes form mixed particles, APol-detergent and IMP-APol-detergent, respectively, according to the regular mixing model. Diluting IMP-APol-detergent complexes below the critical micellar concentration (CMC) of the detergent triggers the dispersion of detergent molecules as monomers, a process characterized by an enthalpy of demicellization. The enthalpy of APol <--> detergent exchange on the hydrophobic surface of IMPs is negligibly small, an indication of the similarity of the molecular interactions of IMPs with the two types of amphiphiles. The enhanced stability against dilution of IMP-APol complexes, compared to IMP-detergent ones, originates from the difference in entropy gain achieved upon release in water of a few APol molecules (in the case of IMP-APol complexes) or several hundred detergent molecules (in the case of IMP-detergent complexes). The data account both for the stability of IMP-APols complexes in the absence of detergent and for the ease with which detergents displace APols from the surface of proteins.

Complexation of integral membrane proteins by phosphorylcholine-based amphipols.

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b(6)f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols ( approximately 22 kDa) of low dispersity in length, containing approximately 30 mol% octyl side groups, approximately 35 mol% PC-groups, and approximately 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH> or =5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.

Prediction of the positioning of the seven transmembrane alpha-helices of bacteriorhodopsin. A molecular simulation study.

We have applied a search strategy for determining the optimal packing of protein secondary structure elements to the rotational positioning of the seven transmembrane helices of bacteriorhodopsin. The search is based on the assumption that the relative orientations of the helices within the bundle are conditioned principally by inter-helix side-chain interactions and that the extra-helical parts of the protein have only a minor influence on the bundle conformation. Our approach performs conformational energy optimization using a predetermined set of side-chain rotamers and appropriate methods for sampling the conformational space of peptide fragments with fixed backbone geometries. The final solution obtained for bacteriorhodopsin places each of the seven helices to a precision of a few degrees in rotation around the helical axis and to a few tenths of an ångström in translation along the helical axis with respect to the best experimental structure obtained by electron diffraction, except for helix D, where our results support the suggestion that this helix should be displaced along its axis toward its N terminus. The perspectives of such an approach for the determination of the structures of other transmembrane helical bundles are discussed.

Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process.

Possible steps in the folding of bacteriorhodopsin are revealed by studying the refolding and interaction of two fragments of the molecule reconstituted in lipid vesicles. (1) Two denatured bacteriorhodopsin fragments have been purified starting from chymotryptically cleaved bacteriorhodopsin. Cleaved bacteriorhodopsin has been renatured from a mixture of the fragments in Halobacterium lipids/retinal/dodecyl sulfate solution following removal of dodecyl sulfate by precipitation with potassium. The renatured molecules have the same absorption spectrum and extinction coefficient as native cleaved bacteriorhodopsin. They are integrated into small lipid vesicles as a mixture of monomers and aggregates. Extended lattices form during the partial dehydration process used to orient samples for X-ray and neutron crystallography. (2) Correct refolding of cleaved bacterioopsin occurs upon renaturation in the absence of retinal. Regeneration of the chromophore and reformation of the purple membrane lattice are observed following subsequent addition of all-trans retinal. (3) The two chymotryptic fragments have been reinserted separately into lipid vesicles and refolded in the absence of retinal. Circular dichroism spectra of the polypeptide backbone transitions indicate that they have regained a highly alpha-helical structure. The kinetics of chromophore regeneration following reassociation have been studied by absorption spectroscopy. Upon vesicle fusion, the refolded fragments first reassociate, then bind retinal and finally regenerate cleaved bacteriorhodopsin. The complex formed in the absence of retinal is kinetically indistinguishable from cleaved bacterioopsin. The refolded fragments in lipid vesicles are stable for months, both as separate entities and after reassociation. These observations provide further evidence that the native folded structure of bacteriorhodopsin lies at a free energy minimum. They are interpreted in terms of a two-stage folding mechanism for membrane proteins in which stable transmembrane helices are first formed. They subsequently pack without major rearrangement to produce the tertiary structure.

Tertiary structure of bacteriorhodopsin. Positions and orientations of helices A and B in the structural map determined by neutron diffraction.

Positions and rotations of two helices in the tertiary structure of bacteriorhodopsin have been studied by neutron diffraction using reconstituted, hybrid purple membrane samples. Purple membrane was biosynthetically 2H-labeled at non-exchangeable hydrogen positions of leucine and tryptophan residues. Two chymotryptic fragments were purified, encompassing either the first two or the last five of the seven putative transmembrane segments identified in the amino acid sequence of bacteriorhodopsin. The 2H-labeled fragments, diluted to variable extents with the identical, unlabeled fragment, were mixed with their unlabeled counterpart; bacteriorhodopsin was then renatured and reconstituted. The crystalline purple membrane samples thus obtained contained hybrid bacteriorhodopsin molecules in which certain transmembrane segments had been selectively 2H-labeled to various degrees. Neutron diffraction powder patterns were recorded and analyzed both by calculating difference Fourier maps and by model building. The two analyses yielded consistent results. The first and second transmembrane segments in the sequence correspond to helices 1 and 7 of the three-dimensional structure, respectively. Rotational orientations of these two helices were identified using best fits to the observed diffraction intensities. The data also put restrictions on the position of the third transmembrane segment. These observations are discussed in the context of folding models for bacteriorhodopsin, the environment of the retinal Schiff base, and site-directed mutagenesis experiments.

Rotational orientation of transmembrane alpha-helices in bacteriorhodopsin. A neutron diffraction study.

The rotational orientation of the seven transmembrane alpha-helices (A-G) in bacteriorhodopsin has been investigated by neutron diffraction. The current model of bacteriorhodopsin is based on an electron density map obtained by high-resolution electron microscopy (EM). Assigning helix rotational positions in the EM model depended on fitting large side-chains, mainly aromatic residues, into bulges in the electron density map. For helix D, which contains no aromatic residues, the EM map is more difficult to interpret. For helices A and B, whose position and orientation had been determined previously by neutron diffraction, the positions defined by EM agree within experimental error with these earlier conclusions. The orientation of all seven helices has been examined by using neutron diffraction on bacteriorhodopsin samples with specifically deuterated valine, leucine and tryptophan residues. Experimental peak intensities were compared to those predicted for an extensive set of structural models. The models were generated by (1) rotating all helices around their axis; (2) moving deuterated residues in the extramembrane loops about their probable positions and changing the weight of their contribution to the neutron diffraction pattern; (3) allowing deuterated side-chains to change their conformation. The analysis confirmed exactly the positions previously determined for helices A and B. For an optimal fit to the data to be obtained, the other five helices, including helix D, must lie either at or within 20 degrees of their position in the current EM model. The complementarity of medium-resolution EM, neutron diffraction and model building for the structural study of integral membrane proteins is discussed.

Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata.

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 Mr oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (alpha 2 beta gamma delta) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the delta chains. In agreement with previous work (Cartaud et al., 1980) the H-form appeared as "doublets" of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 +/- 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublets are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the delta chain within the structure. Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified. Tentative assignment of structural details to individual subunits was deduced from an examination of alpha-bungarotoxin-labeled doublets. The alpha subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).

Transmembrane alpha-helix interactions are required for the functional assembly of the Escherichia coli Tol complex.

TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope. TolQ and TolR span the inner membrane with three and with one alpha-helical segments, respectively. The tolQ925 mutation (A177V), located in the third putative transmembrane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts and tolerance towards colicin A but not colicin E1, unlike a null tolQ mutation, which induces tolerance to all group A colicins. Since TolQ is required for colicin A and E1 uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutation might affect an interaction between TolQ and TolR. We therefore searched for suppressor mutations in TolR that would restore cell envelope integrity and colicin A sensitivity to the tolQ925 mutant. Five different tolR alleles were isolated and characterized. Four of these suppressor mutations were found to be clustered in the single putative transmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein. In addition, we isolated a spontaneous intragenic suppressor localized in the first transmembrane helix of TolQ (TolQ-I). These observations strongly suggest that TolR and TolQ interact via their transmembrane segments. Sequence analysis indicates that Ala177 lies on the alpha-helix face of TolQ-III that, according to its composition and evolutionary conservation, is the most likely to be involved in protein/protein interaction. Energy minimization of atomic models of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a direct steric hindrance of this residue with neighboring transmembrane segments, and that suppressor mutations may alleviate this effect either directly or indirectly, e.g. by affecting the stability of conformational equilibrium of the transmembrane region of the complex.

Engineering membrane proteins.

Much of the research on integral membrane proteins mirrors that on soluble proteins; however, membrane protein engineering also has its own ends and means, many of which take advantage of the peculiar situation of membrane proteins, whose chains are distributed between one lipidic and two aqueous phases. Extramembrane loops have been shortened, cut, or elongated with segments forming proteolytic cleavage sites, foreign epitopes, extra transmembrane segments, or even whole proteins, with the aim of facilitating purification, biochemical/biophysical studies, or crystallogenesis. Transmembrane alpha-helices have been deleted, duplicated, exchanged, transported into a foreign context or replaced with synthetic peptides, in order to both understand their integration into, and assembly in, the membrane and unravel their functional role. Insertion of cysteine residues has been the basis for a great diversity of experiments, ranging from the exploration of secondary, tertiary and quaternary structures of the transmembrane region to the creation of anchoring points for reporter molecules. Chemical engineering--the synthesis of protein fragments or even of whole proteins--offers particularly exciting new prospects, given the small size of folding domains in alpha-helical membrane proteins. Membrane protein engineering is rapidly developing its own agenda of questions and tool chest of techniques.

Scanning transmission electron microscopy study of the molecular mass of amphipol/cytochrome b6f complexes.

The composition and mass of complexes between Chlamydomonas reinhardtii cytochrome b6f and low molecular mass amphipathic polymers ('amphipols') have been studied using biochemical analysis and scanning transmission electron microscopy at liquid helium temperature (cryo-STEM). Cytochrome b6f was trapped by amphipols either under its native 14-meric state or as a delipidated, lighter form. A good consistency was observed between the masses of either form calculated from their biochemical composition and those determined by cryo-STEM. These data show that association with amphipols preserved the original original state of the protein in detergent solution. Complexation with amphipols appears to facilitate preparation of the samples and mass determination by cryo-STEM as compared to conventional solubilization with detergents.

Stabilization of integral membrane proteins in aqueous solution using fluorinated surfactants.

Surfactants carrying either a hydrocarbon or a fluorocarbon alkyl chain have been synthesized. The polar head was either tris(hydroxymethyl)acrylamidomethane (THAM), telomerized THAM, or a glycosylated THAM moiety. The aqueous solubility of some of these molecules was increased by oxidizing to a sulfoxide the thioether function that associates their hydrophobic and hydrophilic moieties. In all cases, the critical micellar concentration was principally determined by the length and chemical nature of the alkyl chain. The usefulness of these surfactants in handling integral membrane proteins in solution has been examined using as test materials chloroplast thylakoid membranes and the photosynthetic complex cytochrome b6f. In keeping with earlier observations in other systems, none of the fluorinated surfactants was able to solubilize thylakoid membranes. Transfer to a solution of fluorinated surfactant of b6f complexes that had been solubilized and purified in the presence of a classical detergent usually resulted in aggregation and precipitation of the protein, while most homologous molecules with hydrocarbon chains did keep the b6f complex soluble. Two of the fluorinated surfactants, however, proved able to maintain the b6f complex water-soluble, intact, and enzymatically active. Because of their limited affinity for lipid alkyl chains and other hydrocarbon surfaces, fluorinated surfactants appear as potentially interesting tools for the study of membrane proteins that do not stand well exposure to classical detergents.

Synthesis and preliminary assessments of ethyl-terminated perfluoroalkyl nonionic surfactants derived from tris(hydroxymethyl)acrylamidomethane.

We describe the synthesis and preliminary physicochemical and biological assessments of a new class of nonionic hybrid hydrofluoro amphiphiles derived from tris(hydroxymethyl)aminomethane (THAM). The synthesis of the hydrophobic tail of these amphiphiles is based on the preparation of an asymmetrical hydrofluorocarbon derivative containing an ethyl segment, a fluorocarbon core, and an ethyl thiol moiety. This molecule led to either THAM galactosylated monoadducts or telomers. These amphiphiles exhibit neither detergency toward cell membranes nor membrane protein denaturation.

Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.