Methods for Studying Interactions of Detergents and Lipids with α‐Helical and β‐Barrel Integral Membrane Proteins

S. Saif Hasan1, Danas Baniulis2, Eiki Yamashita3, Mariya V. Zhalnina1, Stanislav D. Zakharov4, Jason T. Stofleth5, William A. Cramer1

1 Department of Biological Sciences, Purdue University, West Lafayette, Indiana, 2 Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai, Kaunas Region, 3 Institute for Protein Research, Osaka University, Suita, Osaka, 4 Institute of Basic Problems of Biology, Russian Academy of Sciences, Pushchino, Moscow Region, 5 Department of Chemistry and Biochemistry, University of California San Diego, San Diego
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.7
DOI:  10.1002/0471140864.ps2907s74
Online Posting Date:  November, 2013
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Abstract

Methods for studying interactions of protein with lipids and detergents are described for representatives of two major classes of membrane proteins: (1) the α‐helical hetero‐oligomeric integral cytochrome b6f complex of oxygenic photosynthesis from cyanobacteria, and (2) the outer membrane β‐barrel proteins BtuB and OmpF from Gram‐negative Escherichia coli bacteria. Details are presented on the use of detergents for purification and crystallization of the b6f complex as well as a method for lipid exchange. The positions of detergent and lipid molecules, which define eight potential lipid‐binding sites in the b6f complex, are described. Differences in detergent strategies for isolation and crystallization of β‐barrel proteins relative to those for oligomeric helical membrane proteins are discussed, and purification and assessment of protein quality by circular dichroism (CD) is presented. Curr. Protoc. Protein Sci. 74:29.7.1‐29.7.30. © 2013 by John Wiley & Sons, Inc.

Keywords: BtuB; vitamin B12 receptor; cytochrome b6f complex; OmpF porin

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Screening Detergents to Determine Membrane Protein Extraction Efficiency
  • Basic Protocol 2: Exchanging Membrane Protein Native Lipids
  • Basic Protocol 3: Extraction and Purification of β‐Barrel Proteins BtuB and OmpF from the Outer Membrane of E. coli
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Screening Detergents to Determine Membrane Protein Extraction Efficiency

  Materials
  • Nostoc PCC 7120 cells (ATCC)
  • BG11 medium (see recipe),
  • CO 2 gas
  • Cell breakage buffer (see recipe)
  • Osmotic shock buffer (see recipe)
  • Membrane wash buffer (see recipe)
  • TNE buffer 1 (see recipe)
  • Acetone
  • Extraction buffer (see recipe)
  • 10% (w/v) UDM‐Sol (see ) in extraction buffer
  • 25 mM decylplastoquinone (dPQ; Sigma‐Aldrich) in 100% ethanol in a cuvette sealed with a rubber lid
  • Carbon‐coated platinum catalyst (Sigma‐Aldrich)
  • Hydrogen gas
  • Cyt b 6f activity assay buffer (see recipe)
  • Ethanol
  • 13‐liter carboys
  • Gas regulators, glass and rubber tubing, and in‐line barometer (Cole‐Palmer) for CO 2 supply
  • Spectrophotometer with thermostat and stirring control (Cary 4000 from Varian or equivalent)
  • 3‐ml glass cuvette and small magnetic bead for stirring
  • 1‐ml UV‐transparent cuvette
  • Pellicon tangential flow filter (Millipore)
  • 40‐ml homogenizer with glass jacket and Teflon pestle, 4°C
  • 300‐ml glass beakers
  • French pressure cell, 4°C
  • 26.3‐ml ultracentrifuge bottles (Beckman‐Coulter)
  • Ultracentrifuge with Ti‐70 and TLA‐100.3 rotors (Beckman‐Coulter)
  • Paint brush
  • Electric drill
  • 1.5‐ml polyallomer microcentrifuge tubes (Beckman‐Coulter)
  • 1‐ml bullet‐shaped interior cell vial with rubber lid
  • 1‐ and 1.5‐in. syringe needles
  • Parafilm
  • JA‐10 rotor (Beckman‐Coulter) with 500‐ml Nalgene bottles
NOTE: Unless otherwise noted, all centrifugations are carried out at 4°C.

Basic Protocol 2: Exchanging Membrane Protein Native Lipids

  Materials
  • Propyl agarose chromatography resin (Sigma‐Aldrich)
  • Chromatography wash buffer (see recipe)
  • Membrane suspension containing Cyt b 6f complex from cyanobacterial membranes (see protocol 1)
  • 10% (w/v) UDM‐Sol (see recipe) in TNE/sucrose buffer
  • Pulverized ammonium sulfate
  • Elution buffer (see recipe)
  • Protein buffer for sucrose density gradient centrifugation: TNE buffer 1 (see recipe) with 0.05% UDM‐Sol (prepare on day of experiment)
  • 10% and 32% (w/v) sucrose gradient solutions (see recipe)
  • Desired lipids, e.g., neutral dioleoylphosphatidylcholine and acidic dioleoylphosphatidylglycerol (Avanti Polar Lipids)
  • Gaseous nitrogen
  • Desiccator
  • TNS buffer (see recipe)
  • 10% (w/v) UDM‐Ana (see recipe) in TNS buffer
  • 20‐ml, 1‐cm‐diameter glass chromatography column
  • Peristaltic pump for chromatography
  • 5‐ml fraction collection tubes
  • 100‐ and 300‐ml glass beakers
  • 20‐ml syringe and 0.45‐µm syringe filter
  • 100‐kDa nominal molecular weight cut‐off (MWCO) protein concentrators (Millipore)
  • SG15 gradient maker (Hoefer)
  • SW41‐Ti rotor with canisters and tubes (Beckmann‐Coulter), 4 °C
  • 15‐ml polycarbonate test tubes (Thermo Scientific Nunc)
  • 16 × 150‐mm glass test tubes (Sigma‐Aldrich)
  • Parafilm
  • Liquid nitrogen
  • Sonicator with water bath
NOTE: Unless otherwise noted, all centrifugations are carried out at 4°C.

Basic Protocol 3: Extraction and Purification of β‐Barrel Proteins BtuB and OmpF from the Outer Membrane of E. coli

  Materials
  • LB medium
  • 0.5 M ampicillin or 0.25 M kanamycin (for BtuB or OmpF, respectively)
  • E. coli strain TNE012 (pJC3) AmpR or MH225 (pPR272) KanR (for BtuB or OmpF, respectively)
  • Buffer A: 50 mM Tris, pH 8.0, with 2 mM EDTA
  • 100× protease inhibitor solution (see recipe)
  • 2 M MgSO 4
  • DNase 1
  • Triton X‐100
  • 1.5% or 3.0% (w/v) β‐octylglucoside in buffer A (for BtuB or OmpF, respectively)
  • 1% (w/v) β‐octylglucoside in buffer A
  • 0.1% (w/v) lauryldiamine‐N‐oxide (LDAO, 1‐dodecanamine‐N,N‐dimethyloxide) or 0.8% (w/v) N‐octylpolyoxyethylene (octyl‐POE) in buffer A (for BtuB or OmpF, respectively)
  • LiCl
  • 0.1% LDAO in TNE buffer 2 (see recipe), degassed
  • Nitrogen gas
  • 18 × 150−mm culture tubes with caps
  • 2‐liter flasks
  • Sterile toothpicks
  • Incubator shaker (e.g., Innova 4430, New Brunswick Scientific)
  • Spectrophotometer with 1‐cm cuvette (for determining cell density)
  • Avanti J‐E centrifuge with JA‐10 rotor and appropriate bottles (Beckman‐Coulter) or equivalent
  • Teflon homogenizer with glass jacket
  • Continuous‐flow French press
  • JA‐25.5 rotor and appropriate bottles (Beckman‐Coulter) or equivalent
  • Optima LE‐80K centrifuge with Ti70 rotor and appropriate bottles (Beckman‐Coulter) or equivalent
  • Anion‐exchange column(s):
    • DEAE‐Sepharose and Q‐agarose columns (HiPrep FF 16/10, GE Healthcare Life Sciences; for BtuB)
    • FPLC system (Acta‐FPLC, GE Healthcare Life Sciences; for OmpF)
  • CentriPrep 50 concentrators (Millipore)
  • Superdex 200 column (FF 10/300, GE Healthcare Life Sciences)
  • Cary Bio 300 UV Vis spectrophotometer (Varian) and quartz cuvettes with 1‐mm or 1‐cm optical path length
  • CD spectrophotometer (e.g., Charascan, Applied Photophysics) and demountable quartz cuvette with 0.1‐mm path length (Starna Cells or Hellma Cells)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
CAUTION: PMSF is highly toxic. Wear gloves and work in a chemical fume hood when using PMSF.NOTE: Unless otherwise noted, all centrifugations are carried out at 4°C.
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Figures

Videos

Literature Cited

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