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The Journal of Membrane Biology

, Volume 247, Issue 9–10, pp 925–940 | Cite as

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

  • Yann Ferrandez
  • Manuela Dezi
  • Mickael Bosco
  • Agathe Urvoas
  • Marie Valerio-Lepiniec
  • Christel Le Bon
  • Fabrice Giusti
  • Isabelle Broutin
  • Grégory Durand
  • Ange Polidori
  • Jean-Luc Popot
  • Martin PicardEmail author
  • Philippe Minard
Article

Abstract

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for “artificial alpha repeat protein”) have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal “Velcro” to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

Keywords

HEAT repeat protein Protein design Phage display Membrane protein Amphipols Immobilization 

Abbreviations

2XTY

E. coli rich media

A8-35

A specific type of poly(acrylic acid)-based amphipol

APol

Amphipol

BAPol

Biotinylated A8-35

BNAPol

Biotinylated non-ionic amphipol

BR

Bacteriorhodopsin

cmc

Critical micellar concentration

DDM

Dodecyl-β-d-maltoside

\(\overline{DP}_{n}\)

Number-average degree of polymerization

EDTA

Ethylene diamine tetraacetic acid

EM

Electron microscopy

His-tag

Hexahistidine tag

IPTG

Isopropyl-β-d-1-thiogalactopyranoside

MD

Molecular dynamics

\(\overline{M}_{n}\)

Number-average molar mass

MP

Membrane protein

MW

Molecular weight

MWCO

MW cut-off

NAPol

Non-ionic amphipol

NMR

Nuclear magnetic resonance

NTA

Nitrilotriacetic acid

OD600nm

Optical density measured at 600 nm

βOG

n-octyl-β-d-glucopyranoside

PBS

Phosphate buffer saline

PEG

Polyethylene glycol

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEC

Size-exclusion chromatography

TBS

Tris-buffered saline

TBST

Tris-buffered saline supplemented with Tween 20 (w/v)

Tris

Tris(hydroxymethyl)aminomethane

Notes

Acknowledgments

Particular thanks are due to J.D. Perlmutter and J.N. Sachs for communication of a molecular dynamics model of an A8-35 particle. This research was supported by ANR-2010-BLAN-1535, by the French Centre National de la Recherche Scientifique (CNRS), by Université Paris-5 Paris Descartes, by Université Paris-7 Denis Diderot, and by grant “DYNAMO”, ANR-11-LABX-0011-01 from the French “Initiative d’Excellence” program.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Yann Ferrandez
    • 1
  • Manuela Dezi
    • 2
  • Mickael Bosco
    • 3
    • 4
  • Agathe Urvoas
    • 1
  • Marie Valerio-Lepiniec
    • 1
  • Christel Le Bon
    • 5
  • Fabrice Giusti
    • 5
  • Isabelle Broutin
    • 2
  • Grégory Durand
    • 3
    • 4
  • Ange Polidori
    • 3
    • 4
  • Jean-Luc Popot
    • 5
  • Martin Picard
    • 2
    Email author
  • Philippe Minard
    • 1
  1. 1.Laboratoire de Modélisation et Ingénierie des ProtéinesIBBMC UMR 8619, CNRS/Université Paris SudOrsayFrance
  2. 2.Laboratoire de Cristallographie et RMN Biologiques, Faculté de Pharmacie, UMR 8015CNRS/Université Paris DescartesParis Cedex 06France
  3. 3.Equipe Chimie Bioorganique et Systèmes AmphiphilesUniversité d’AvignonAvignonFrance
  4. 4.Institut des Biomolécules Max Mousseron (UMR 5247)Montpellier Cedex 05France
  5. 5.Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099Institut de Biologie Physico-Chimique (FRC 550), Centre National de la Recherche Scientifique/Université Paris-7ParisFrance

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