The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 909–924 | Cite as

Synthesis, Characterization and Applications of a Perdeuterated Amphipol

  • Fabrice Giusti
  • Jutta Rieger
  • Laurent J. Catoire
  • Shuo Qian
  • Antonio N. Calabrese
  • Thomas G. Watkinson
  • Marina Casiraghi
  • Sheena E. Radford
  • Alison E. Ashcroft
  • Jean-Luc Popot
Article

Abstract

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization–ion mobility spectrometry–mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120 % D2O. In 1H-1H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6 % of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.

Keywords

Amphipol A8-35 Deuteration Mass spectrometry NMR 

Abbreviations

1D, 2D, 3D

One-, two- and three-dimensional, respectively

A8-35

Sodium poly(acrylate)-based amphipol with a weight-average molar mass close to 8 kDa and containing 35 % of free carboxylate

A8-75

Sodium poly(acrylate)-based amphipol with a weight-average molar mass close to 8 kDa and containing 75 % of free carboxylate

AA

Acrylic acid

AAd4

Acrylic acid-d4

AIBN

2,2′-Azoisobutyronitrile

APol

Amphipol

AUC

Analytical ultracentrifugation

CMP

Neutron scattering contrast match point

Ct

Transfer constant

DAPol

A8-35 with perdeuterated side chains

DCI

Dicyclohexylcarbodiimide

DCU

N,N-Dicyclohexylurea

\(\overline{{DP_{n} }}\)

Average degree of polymerization in number

dV

Differential viscometry

Đ

Molar mass dispersity

ESI

Electrospray ionization

HAPol

Hydrogenated A8-35

HSQC

Heteronuclear single quantum correlation

HOBt

1-N-Hydroxybenzotriazole

IMS

Ion mobility spectrometry

INS

Inelastic neutron scattering

LTB4

Leukotriene B4

\(\overline{{M_{n} }}\)

Number-average molar mass

mQ water

Water purified on a A10 advantage millipore system

MS

Mass spectrometry

\(\overline{{M_{w} }}\)

Weight-average molar mass

NAPol

Non-ionic amphipol

NMP

N-Methylpyrrolidone

NOE

Nuclear Overhauser effect

NOESY

NOE spectroscopy

PAA

Poly(acrylic acid)

perDAPol

Perdeuterated A8-35

PEO

Poly(ethylene oxide)

PS

Polystyrene

RI

Refractive index

RS

Stokes radius

SANS

Small-angle neutron scattering

SAPol

Sulfonated amphipol derived from A8-75

SEC

Size-exclusion chromatography

TA

Transfer agent

TGA

Thioglycolic acid

THF

Tetrahydrofuran

TMSCHN2

Trimethylsilyldiazomethane

WHH

Width at half-height

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Fabrice Giusti
    • 1
  • Jutta Rieger
    • 2
    • 3
  • Laurent J. Catoire
    • 1
  • Shuo Qian
    • 4
  • Antonio N. Calabrese
    • 5
  • Thomas G. Watkinson
    • 5
  • Marina Casiraghi
    • 1
  • Sheena E. Radford
    • 5
  • Alison E. Ashcroft
    • 5
  • Jean-Luc Popot
    • 1
  1. 1.Laboratoire de Physico-Chimie Moléculaire des Membranes Biologiques, UMR 7099, Institut de Biologie Physico-Chimique (FRC 550)Centre National de la Recherche Scientifique and Université Paris-7ParisFrance
  2. 2.UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des PolymèresSorbonne Universités, UPMC Univ Paris 06ParisFrance
  3. 3.UMR 8232, Institut Parisien de Chimie Moléculaire (IPCM), Equipe Chimie des PolymèresCNRSParisFrance
  4. 4.Center for Structural Molecular Biology and Biology and Soft Matter DivisionOak Ridge National LaboratoryOak RidgeUSA
  5. 5.Astbury Centre for Structural Molecular Biology, School of Molecular and Cellular BiologyUniversity of LeedsLeedsUK

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