Fluorescence Quenching Methods to Study Lipid‐Protein Interactions

Joanne Carney1, J. Malcolm East1, Sanjay Mall1, Phedra Marius1, Andrew M. Powl1, J. Neville Wright1, Anthony G. Lee1

1 University of Southampton, Southampton
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.12
DOI:  10.1002/0471142301.ps1912s45
Online Posting Date:  September, 2006
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Abstract

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp‐containing intrinsic membrane protein with bromine‐containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine‐containing phospholipids from phospholipids containing carbon‐carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine‐containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.

Keywords: Lipid‐protein interactions; reconstitution; membrane proteins; fluorescence; phospholipid synthesis; bromination

     
 
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Table of Contents

  • Basic Protocol 1: Reconstitution of A Membrane Protein into DOPC and BrPC and Measurement of Fluorescence Quenching Efficiency
  • Basic Protocol 2: Measurement of the Relative Lipid‐Binding Constant of A Membrane Protein Using Fluorescence Quenching
  • Support Protocol 1: Preparation of Brominated Lipid
  • Support Protocol 2: Analysis of Brominated Phospholipid by Mass Spectrometry
  • Support Protocol 3: Purification of Potassium Cholate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Reconstitution of A Membrane Protein into DOPC and BrPC and Measurement of Fluorescence Quenching Efficiency

  Materials
  • 25 mg/ml dioleoylphosphatidylcholine (DOPC; Avanti Polar Lipids) in chloroform
  • 25 mg/ml di(9,10‐dibromostearoyl)phosphatidylcholine (BrPC; protocol 3) in chloroform
  • Oxygen‐free nitrogen
  • Reconstitution buffer: HEPES/EGTA buffer (see recipe) containing 15 mM potassium cholate purified as in protocol 5
  • KcsA (purified as His‐tagged protein in Mega‐9 or dodecylmaltoside, eluting from metal‐chelate column with imidazole; Doyle et al., ; Williamson et al., ) at 2.5 mg protein/ml
  • HEPES/EGTA buffer (see recipe)
  • 10‐ml screw‐cap glass scintillation vials, 1‐cm diameter
  • Vacuum desiccator connected to vacuum pump
  • 50°C water bath
  • Retort stand and clamp
  • Ultrasonication bath (model FB11010, Fisher Scientific, or equivalent)
  • 4‐ml quartz fluorescence cell
  • Fluorimeter

Basic Protocol 2: Measurement of the Relative Lipid‐Binding Constant of A Membrane Protein Using Fluorescence Quenching

  Materials
  • 25 mg/ml dioleoylphosphatidylcholine (DOPC; Avanti Polar Lipids) in chloroform
  • 25 mg/ml di(9,10‐dibromostearoyl)phosphatidylcholine (BrPC; protocol 3) in chloroform
  • Oxygen‐free nitrogen
  • Reconstitution buffer: HEPES/EGTA buffer (see recipe) containing 15 mM potassium cholate purified as in protocol 5
  • KcsA (purified as His‐tagged protein in Mega‐9 or dodecylmaltoside, eluting from metal‐chelate column with imidazole; Doyle et al., ; Williamson et al., ) at 2.5 mg protein/ml
  • HEPES/EGTA buffer (see recipe)
  • 25 mg/ml dioleoylphosphatidylethanolamine (DOPE; Avanti Polar Lipids) in chloroform
  • 10‐ml screw‐cap glass scintillation vials, 1‐cm diameter
  • Vacuum desiccator connected to vacuum pump
  • 50°C shaking water bath
  • Retort stand and clamp
  • Ultrasonication bath (model FB11010, Fisher Scientific, or equivalent)
  • 4‐ml quartz fluorescence cell
  • Fluorimeter
  • SigmaPlot (Sigma) or equivalent data‐fitting program

Support Protocol 1: Preparation of Brominated Lipid

  Materials
  • 25 mg/ml dioleoylphosphatidylcholine (DOPC; Avanti Polar Lipids) in chloroform
  • Bromine, liquid (Br 2)
  • 95:35:6 (v/v/v) chloroform/methanol/water or 14:6:1 (v/v/v) chloroform/methanol/25% (w/v) ammonia
  • 10‐ml round‐bottom flask with ground‐glass stopper
  • Retort stand
  • Glass tank with cover, large enough to hold 3 × 7 – cm TLC plate and second chromatography tank of the same size containing a few crystals of solid iodine (I 2)
  • 3 × 7–cm thin‐layer chromatography (TLC) plate (plastic‐backed silica gel G plate without fluorophore; Whatman 4410‐221) cut from a 20 × 20–cm sheet
  • Hot air dryer

Support Protocol 2: Analysis of Brominated Phospholipid by Mass Spectrometry

  Materials
  • 1 mg/ml di(9,10‐dibromostearoyl)phosphatidylcholine (BrPC; protocol 3) in chloroform
  • 7:2:1 (v/v/v) methanol/chloroform/water
  • 2 M HCl
  • 2 mg/ml NaI in 1:1 propan‐2‐ol/H 2O
  • ∼2‐ml glass test tubes
  • 1‐ml gastight syringe with Teflon luer‐lock connector (Hamilton P/N 81322)
  • Quattro II triple quadrupole mass spectrometer with electrospray probe (Waters)
  • PEEK tubing (0.5 m length, 1.6 mm o.d., 0.12 mm i.d.)
  • PEEK Fingertight HPLC fittings (Upchurch)
  • PEEK Quick Connect luer adapter (Upchurch, P/N P‐659)
  • Additional reagents and equipment for electrospray ionization mass spectrometry (unit 16.8)

Support Protocol 3: Purification of Potassium Cholate

  Materials
  • Cholic acid
  • Methanol, pure‐grade
  • Potassium hydroxide
  • Diethyl ether
  • 1‐liter Erlenmeyer flask
  • 40°C water bath
  • Large sintered‐glass filter funnel
  • Glass beaker
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Figures

Videos

Literature Cited

Literature Cited
  Alvis, S.J., Williamson, I.M., East, J.M., and Lee, A.G. 2003. Interactions of anionic phospholipids and phosphatidylethanolamines with the potassium channel KcsA. Biophys. J. 85:1‐11.
   Bergelson, L.D. 1980. Lipid Biochemical Preparations. Elsevier/North Holland, Amsterdam.
   Caffrey, M. and Feigenson, G.W. 1981. Fluorescence quenching in model membranes. 3. Relationship between calcium adenosinetriphosphatase enzyme activity and the affinity of the protein for phosphatidylcholines with different acyl chain characteristics. Biochemistry 20:1949‐1961.
   Doyle, D.A., Cabral, J.M., Pfuetzner, R.A., Kuo, A., Gulbis, J.M., Cohen, S.L., Chait, B.T., and Mackinnon, R. 1998. The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science 280:69‐77.
   East, J.M. and Lee, A.G. 1982. Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid. Biochemistry 21:4144‐4151.
   East, J.M., Melville, D., and Lee, A.G. 1985. Exchange rates and numbers of annular lipids for the calcium and magnesium ion dependent adenosinetriphosphatase. Biochemistry 24:2615‐2623.
   Froud, R.J., East, J.M., Rooney, E.K., and Lee, A.G. 1986. Binding of long‐chain alkyl derivatives to lipid bilayers and to (Ca2+−Mg2+)‐ATPase. Biochemistry 25:7535‐7544.
   Lee, A.G. 2004. How lipids affect the activities of integral membrane proteins. Biochim. Biophys. Acta 1666:62‐87.
   London, E. and Feigenson, G.W. 1981. Fluorescence quenching in model membranes. 2. Determination of local lipid environment of the calcium adenosinetriphosphatase from sarcoplasmic reticulum. Biochemistry 20:1939‐1948.
   Mall, S., Sharma, R.P., East, J.M., and Lee, A.G. 1998. Lipid‐protein interactions in the membrane: Studies with model peptides. Faraday Discuss. 111:127‐136.
   Marsh, D. and Horvath, L.I. 1998. Structure, dynamics and composition of the lipid‐protein interface: Perspectives from spin‐labelling. Biochim. Biophys. Acta 1376:267‐296.
   O'Keeffe, A.H., East, J.M., and Lee, A.G. 2000. Selectivity in lipid binding to the bacterial outer membrane protein OmpF. Biophys. J. 79:2066‐2074.
   Pilot, J.D., East, J.M., and Lee, A.G. 2001. Effects of bilayer thickness on the activity of diacylglycerol kinase of Escherichia coli. Biochemistry 40:8188‐8195.
   Powl, A.M., East, J.M., and Lee, A.G. 2003. Lipid‐protein interactions studied by introduction of a tryptophan residue: The mechanosensitive channel MscL. Biochemistry 42:14306‐14317.
   Powl, A.M., East, J.M., and Lee, A.G. 2005. Heterogeneity in the binding of lipid molecules to the surface of a membrane protein: Hot‐spots for anionic lipids on the mechanosensitive channel of large conductance MscL and effects on conformation. Biochemistry 44:5873‐5883.
   Simmonds, A.C., East, J.M., Jones, O.T., Rooney, E.K., McWhirter, J., and Lee, A.G. 1982. Annular and non‐annular binding sites on the (Ca2+ + Mg2+)‐ATPase. Biochim. Biophys. Acta 693:398‐406.
   Williamson, I.M., Alvis, S.J., East, J.M., and Lee, A.G. 2002. Interactions of phospholipids with the potassium channel KcsA. Biophys. J. 83:2026‐2038.
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