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Labeling and Functionalizing Amphipols for Biological Applications

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Abstract

Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.

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Abbreviations

A8-35:

Poly(sodium acrylate) based amphipol comprising 35 % of free carboxylate, 25 % of octyl chains, 40 % of isopropyl groups, and whose number-average molar mass is ~4.3 kDa

A8-75:

Poly(sodium acrylate) based amphipol comprising 75 % of free carboxylate, 25 % of octylchains, and whose number-average molar mass is ~4 kDa

APol:

Amphipol

AUC:

Analytical ultracentrifugation

BAPol:

Biotinylated A8-35

Ð:

Molar mass dispersity

DAPol:

A8-35 with deuterated octylamine and isopropylamine side chains

DCI:

N,N′-dicyclohexylcarbodiimide

DCU:

Dicyclohexylurea

DTT:

Dithiothreitol

EDC:

Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride

FAPol:

Fluorescently labeled A8-35

FAPolAF647 :

Alexa Fluor 647-labeled A8-35

FAPolNBD :

Nitrobenzoxadiazole-labeled A8-35

FAPolrhod :

Rhodamine-labeled A8-35

FRET:

Förster resonance energy transfer

HAPol:

Hydrogenated A8-35

HMPA:

Hydrophobically modified poly(acrylic acid)

HMPAS:

HMPA synthesis

His6PEG:

N-(penta(histidyl)histidinamide)-8-amino-3,6-dioxa-octanamide

His-tag:

Hexahistidine tag

HistAPol:

Hexahistidine tag-carrying A8-35

HOBt:

1-N-hydroxybenzotriazole

IMAC:

Immobilized metal ion affinity chromatography

ImidAPol:

Imidazole-carrying A8-35

INS:

Inelastic neutron scattering

〈M n〉:

Number-average molar mass

MP:

Membrane protein

NBD:

7-Nitrobenz-2-oxa-1,3-diazol-4-yl

NHS:

N-hydroxysuccinimide

NOE:

Nuclear Overhauser effect

NTA:

Nitrilotriacetic acid

ODN:

Oligodeoxynucleotide

OligAPol:

ODN-carrying A8-35

PAA:

Poly(acrylic acid)

perDAPol:

Perdeuterated A8-35

SANS:

Small angle neutron scattering

SAPol:

Sulfonated amphipol derived from A8-75, comprising 40 % of taurine moieties

SPR:

Surface plasmon resonance

TES:

Triethylsilane

TFA:

Trifluoroacetic acid

ThiAPol:

Thiol-carrying APol

Tsv:

Tosvinyl group

UAPol:

Universal amphipol

UAPol-NH2 :

Amine-carrying A8-35

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Acknowledgments

This work was supported by the Center National de la Recherche Scientifique, by Université Paris-7 Denis Diderot, and by U.S. National grant R01AI092129 from the National Institute of Allergy and Infectious Diseases, French Agence Nationale pour la Recherche grant ‘X-Or,’ ANR SVSE5 2010-BLAN-1535, and grant ‘DYNAMO,’ ANR-11-LABX-0011-01 from the French ‘Initiative d’Excellence’ program. CLB has been the beneficiary of funding by the Fondation pour la Recherche Médicale, the Agence Nationale de la Recherche Scientifique (ANR-07-BLAN-0092 ‘Refolding GPCRs’ and SVSE5 2010-BLAN-1535 ‘X-Or’) and the Direction Innovation et Relations avec les Entreprises of the CNRS.

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  1. Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550), CNRS/Université Paris 7, 13 rue Pierre et Marie Curie, 75005, Paris, France

    Christel Le Bon, Jean-Luc Popot & Fabrice Giusti

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  1. Christel Le Bon
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  2. Jean-Luc Popot
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  3. Fabrice Giusti
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Correspondence to Fabrice Giusti.

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Le Bon, C., Popot, JL. & Giusti, F. Labeling and Functionalizing Amphipols for Biological Applications. J Membrane Biol 247, 797–814 (2014). https://doi.org/10.1007/s00232-014-9655-y

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