General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays

Thomas M. Tomasiak1, Bjørn P. Pedersen1, Sarika Chaudhary1, Andrew Rodriguez1, Yaneth Robles Colmanares1, Zygy Roe‐Zurz1, Sobha Thamminana1, Meseret Tessema1, Robert M. Stroud1

1 Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.11
DOI:  10.1002/0471140864.ps2911s77
Online Posting Date:  August, 2014
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Abstract

This unit describes rapid and generally applicable methods to identify conditions that stabilize membrane proteins using temperature‐based denaturation measurements as a proxy for target time‐dependent stability. Recent developments with thiol‐reactive dyes sensitive to the unmasking of cysteine residues upon protein unfolding have allowed for routine application of thermostability assays to systematically evaluate the stability of membrane protein preparations after various purification procedures. Test conditions can include different lipid cocktails, lipid‐detergent micelles, pH, salts, osmolytes, and potential active‐site ligands. Identification and use of conditions that stabilize the structure have proven successful in enabling the structure determination of numerous families of membrane proteins that otherwise were intractable. Curr. Protoc. Protein Sci. 77:29.11.1‐29.11.14 © 2014 by John Wiley & Sons, Inc.

Keywords: thermostability; membrane proteins; dye‐based assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Ramp Method to Measure Membrane Protein Thermostability Using Either CPM Dye or PTS Dye in High‐Throughput Format
  • Basic Protocol 2: Isothermal Method to Determine Membrane Protein Thermostability Using CPM or PTS Dye in qPCR Format
  • Basic Protocol 3: Size‐Exclusion Chromatography (SEC) Profile‐Based Thermostability Measurement of Membrane Proteins
  • Support Protocol 1: Data Processing: Implementing Thermostability Results and Trends
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Ramp Method to Measure Membrane Protein Thermostability Using Either CPM Dye or PTS Dye in High‐Throughput Format

  Materials
  • Isolated membranes for protein purification (see above, Membrane Protein Purification for Structural Studies)
  • Iodoacetamide
  • CPM dye (Life Technologies)
  • Dimethyl sulfoxide (DMSO)
  • PTS dye (Life Technologies)
  • Test buffer (e.g., test conditions varying pH, salt, osmolytes, lipid, and ligands)
  • Method for concentrating protein (e.g., spin concentrator, stir cell)
  • Method for determining protein concentration (e.g., BCA assay, A280 absorbance)
  • qPCR instrument or cuvette fluorometer
  • Aluminum foil
  • Software (GraphPad/Prism from GraphPad Software, Excel/Gnu plot)

Basic Protocol 2: Isothermal Method to Determine Membrane Protein Thermostability Using CPM or PTS Dye in qPCR Format

  Materials
  • Isolated membranes for protein purification
  • Iodoacetamide
  • CPM dye (Life Technologies)
  • Dimethyl sulfoxide (DMSO)
  • Test buffer or ligand solutions (varying pH, salt, lipid, and/or ligands)
  • Method for concentrating protein (e.g., Millipore spin filter, stir cell)
  • Method for determining protein concentration (BCA assay)
  • Aluminum foil
  • Multichannel pipets
  • Temperature‐controlled fluorescent plate reader with selectable wavelength
  • Software (GraphPad/Prism/Gnuplot/Excel)

Basic Protocol 3: Size‐Exclusion Chromatography (SEC) Profile‐Based Thermostability Measurement of Membrane Proteins

  Materials
  • Membrane protein sample
  • Test buffers—including variations of pH, salt, lipids, and ligands
  • FPLC instrument with A 280 optical absorbance detector
  • In‐line fluorometer (for GFP detection assay)
  • Size exclusion chromatography column (e.g., GE Superdex S200, Shodex, GE Sephacryl column, or TSK‐3000)
  • Heat block
  • 0.22‐µm filter or centrifuge filter
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Figures

Videos

Literature Cited

Literature Cited
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