How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins

Synthesis of an oligonucleotide-derivatized amphipol and its use to trap and immobilize membrane proteins

Amphipols (APols) are specially designed amphipathic polymers that stabilize membrane proteins (MPs) in aqueous solutions in the absence of detergent. A8–35, a polyacrylate-based APol, has been grafted with an oligodeoxynucleotide (ODN). The synthesis, purification and properties of the resulting ‘OligAPol’ have been investigated. Grafting was performed by reacting an ODN carrying an amine-terminated arm with the carboxylates of A8–35. The use of OligAPol for trapping MPs and immobilizing them onto solid supports was tested using bacteriorhodopsin (BR) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) as model proteins. BR and OligAPol form water-soluble complexes in which BR remains in its native conformation. Hybridization of the ODN arm with a complementary ODN was not hindered by the assembly of OligAPol into particles, nor by its association with BR. BR/OligAPol and tOmpA/OligAPol complexes could be immobilized onto either magnetic beads or gold nanoparticles grafted with the complementary ODN, as shown by spectroscopic measurements, fluorescence microscopy and the binding of anti-BR and anti-tOmpA antibodies. OligAPols provide a novel, highly versatile approach to tagging MPs, without modifying them chemically nor genetically, for specific, reversible and targetable immobilization, e.g. for nanoscale applications

Membrane proteins in aqueous solutions: from detergents to amphipols

Membrane proteins represent about one third of the proteins encoded in a cell's genome, and, because of their key physiological roles, more than half of drug targets. Detergents are traditionally used to extract proteins from membranes in order to make them amenable to the tools of biochemistry and biophysics. However, detergent-solubilized proteins are generally unstable. This has led to the development of alternative, non-conventional surfactants, such as bicelles, nanodiscs, amphipathic peptides, fluorinated surfactants, and specially designed amphipathic polymers called 'amphipols'. These novel tools, mainly developed over the past 20 years, are revolutionizing handling membrane proteins in vitro for basic and applied research, as well as for such biomedical applications as drug screening or vaccination. This book, written by a specialist of membrane proteins and one of the creators of amphipols, describes the properties and uses of these novel molecules. It opens with general introductions on membrane proteins and their natural environment, detergents, the current status of membrane protein in vitro studies, a broad panorama of non-conventional surfactants and a discussion of their respective advantages and limitations, and the preparation and properties of amphipols and membrane protein/amphipol complexes. Topical chapters cover in vitro folding, cell-free synthesis and stabilization of membrane proteins, and such biophysical and biochemical applications as electron microscopy, Xray diffraction, NMR, optical spectroscopy, mass spectrometry, the whole range of solutions studies, proteomics, and such practical applications as membrane protein immobilization and drug screening and the use of amphipols in vivo for vaccination and drug delivery. Each topical chapter is introduced with a concise, up-to-date overview of how membrane proteins are currently studied using each individual technique, before offering an exhaustive coverage and in-depth discussion of the contribution of amphipols, and concluding with hands-on protocols written by everyday practitioners of each application. In addition to a comprehensive coverage of the properties and uses of non-conventional surfactants, this book therefore also offers a concise, accessible introduction to membrane protein biochemistry and biophysics. It is meant to be used both in basic and applied research laboratories and as a teaching help

Membrane Proteins in Aqueous Solutions

Membrane proteins represent about one third of the proteins encoded in a cell's genome, and, because of their key physiological roles, more than half of drug targets. Detergents are traditionally used to extract proteins from membranes in order to make them amenable to the tools of biochemistry and biophysics. However, detergent-solubilized proteins are generally unstable. This has led to the development of alternative, non-conventional surfactants, such as bicelles, nanodiscs, amphipathic peptides, fluorinated surfactants, and specially designed amphipathic polymers called 'amphipols'. These novel tools, mainly developed over the past 20 years, are revolutionizing handling membrane proteins in vitro for basic and applied research, as well as for such biomedical applications as drug screening or vaccination. This book, written by a specialist of membrane proteins and one of the creators of amphipols, describes the properties and uses of these novel molecules. It opens with general introductions on membrane proteins and their natural environment, detergents, the current status of membrane protein in vitro studies, a broad panorama of non-conventional surfactants and a discussion of their respective advantages and limitations, and the preparation and properties of amphipols and membrane protein/amphipol complexes. Topical chapters cover in vitro folding, cell-free synthesis and stabilization of membrane proteins, and such biophysical and biochemical applications as electron microscopy, Xray diffraction, NMR, optical spectroscopy, mass spectrometry, the whole range of solutions studies, proteomics, and such practical applications as membrane protein immobilization and drug screening and the use of amphipols in vivo for vaccination and drug delivery. Each topical chapter is introduced with a concise, up-to-date overview of how membrane proteins are currently studied using each individual technique, before offering an exhaustive coverage and in-depth discussion of the contribution of amphipols, and concluding with hands-on protocols written by everyday practitioners of each application. In addition to a comprehensive coverage of the properties and uses of non-conventional surfactants, this book therefore also offers a concise, accessible introduction to membrane protein biochemistry and biophysics. It is meant to be used both in basic and applied research laboratories and as a teaching help

Étude structurale et fonctionnelle de deux protéines membranaires, la bactériorhodopsine et le récepteur nicotinique de l'acétylcholine, maintenues en solution aqueuse non détergente par des polymères amphiphiles

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

CONTRIBUTION A L'ETUDE BIOCHIMIQUE DU COMPLEXE CYTOCHROME B 6F DE CHLAMYDOMONAS REINHARDTII (MANIPULATION IN VITRO ET STABILISATION, ORGANISATION SPATIALE DES HEMES ET SPECTROSCOPIE DE LA CHLOROPHYLLE A)

LE CYTOCHROME B 6F DE CHLAMYDOMONAS REINHARDTII A ETE ETUDIE DANS UNE DOUBLE OPTIQUE. D'UNE PART, C'EST UN COMPLEXE TRES FRAGILE, CE QUI EN FAIT UN BON MODELE POUR LE DEVELOPPEMENT DE NOUVEAUX TENSIOACTIFS DESTINES A FACILITER LA MANIPULATION IN VITRO DES PROTEINES MEMBRANAIRES INTEGRALES. LE CYTOCHROME B 6F PRESENTE PAR AILLEURS UN GRAND INTERET PROPRE, CAR IL RESTE LE COMPLEXE PHOTOSYNTHETIQUE DE LOIN LE MOINS BIEN CONNU. NOS MESURES DE DICHROISME LINEAIRE ET CIRCULAIRE SUGGERENT QUE L'ORGANISATION SPATIALE DES HEMES B DES CYTOCHROMES B 6F ET BC 1 (SON HOMOLOGUE MITOCHONDRIAL) SONT SIMILAIRES. CHAQUE DIMERE DE CYTOCHROME B 6F CONTIENT DEUX MOLECULES DE CHLOROPHYLLE A, DONT LA FONCTION ET LA LOCALISATION SONT ENCORE INCONNUES. NOUS MONTRONS L'EXISTENCE DANS NOS PREPARATIONS DE PLUSIEURS POPULATIONS DE CHLOROPHYLLES DE DUREES DE VIE DE FLUORESCENCE DIFFERENTES, CORRESPONDANT PROBABLEMENT A DES MOLECULES LIEES SUR LE SITE NATIF, LIEES SUR UN SITE PARTIELLEMENT DEMASQUE, OU EN SOLUTION DANS LE DETERGENT. L'EXISTENCE D'UN SIGNAL DE DICHROISME CIRCULAIRE EXCITONIQUE SUGGERE QUE LES DEUX CHLOROPHYLLES PRESENTES AU SEIN DE CHAQUE DIMERE SONT SUFFISAMMENT PROCHES L'UNE DE L'AUTRE POUR INTERAGIR ELECTRONIQUEMENT. BIEN QU'INDISPENSABLES A LA SOLUBILISATION ET A LA MANIPULATION DES PROTEINES MEMBRANAIRES EN SOLUTION AQUEUSE, LES DETERGENTS INDUISENT FREQUEMMENT UNE DESTABILISATION POUVANT ALLER JUSQU'A LA DENATURATION. DEUX MECANISMES ENTRENT PROBABLEMENT EN JEU DANS CE PHENOMENE : LA DILUTION DANS LA PHASE MICELLAIRE DE COFACTEURS ESSENTIELS A LA STABILITE DE LA PROTEINE, ET UNE INTRUSION DIRECTE DU DETERGENT DANS LA REGION TRANSMEMBRANAIRE DE CELLE-CI. PARTANT DE L'OBSERVATION QUE LES PERFLUOROALCANES SONT DES MOLECULES RELATIVEMENT RIGIDES ET PEU MISCIBLES AVEC LES HYDROCARBURES, NOUS AVONS MIS AU POINT UN TENSIOACTIF HEMIFLUORE, NON DETERGENT MAIS NEANMOINS CAPABLE DE MAINTENIR EN SOLUTION LE CYTOCHROME B 6F SOUS UNE FORME INTACTE, ACTIVE, MONODISPERSE ET STABLE.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF