Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

Ian Mitchelle S. de Vera1, Mandy E. Blackburn2, Luis Galiano3, Gail E. Fanucci1

1 Department of Chemistry, University of Florida, Gainesville, Florida, 2 Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, 3 Syngenta Crop Protection, Minnetonka, Minnesota
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.17
DOI:  10.1002/0471140864.ps1717s74
Online Posting Date:  November, 2013
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The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron‐electron resonance (DEER) technique. This unit focuses on EPR‐based distance measurements by site‐directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV‐1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance‐distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1‐17.17.29. © 2013 by John Wiley & Sons, Inc.

Keywords: pulsed EPR; DEER; distance measurements; site‐directed spin labeling

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Table of Contents

  • Introduction
  • Nitroxide Spin Labels
  • Selection of Labeling Sites
  • Distance Measurement via DEER
  • Basic Protocol 1: Site‐Directed Spin Labeling of Soluble Proteins
  • Basic Protocol 2: Preparing DEER Samples
  • Basic Protocol 3: Setting Up the EPR Spectrometer and Acquiring DEER Data
  • Basic Protocol 4: Analysis of DEER Data
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Site‐Directed Spin Labeling of Soluble Proteins

  • Soluble protein (e.g., HIV‐1 protease)
  • Labeling buffer (pH 6.5 to 8.0; see discussion below)
  • Nitroxide spin label (SL) with methanethiosulfonate, maleimide, or iodoacetamide moiety : MTSL (Toronto Research Chemicals, cat. no. O875000), MSL (Sigma‐Aldrich, cat. no. 253359), IAP (Sigma‐Aldrich, cat. no. 253421), or IASL (Sigma‐Aldrich, cat. no. 253367), respectively
  • 100% ethanol
  • Tris(2‐carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT)
  • Additional reagents and equipment for liquid chromatography (unit 8.7), dialysis ( appendix 3B), or concentration system for buffer exchange (unit 4.4), and size‐exclusion chromatography

Basic Protocol 2: Preparing DEER Samples

  • Soluble protein (e.g., HIV‐1 protease)
  • Deuterated sodium acetate (D 3‐NaOAc, 99%)
  • Deuterium oxide (D 2O, 99%)
  • Deuterated glycerol (D 8‐glycerol, 99%)
  • Liquid N 2
  • Liquid chromatography or dialysis system (unit 4.4)
  • Concentrator (unit 4.4)
  • 0.2‐ml plastic tubes, sterile
  • Teflon tape
  • 4‐mm quartz EPR tube
  • Syringe fitted with long, narrow Teflon tubing
  • Equipment for handling liquid N 2
  • Additional reagents and equipment for concentration or protein samples (unit 4.4)

Basic Protocol 3: Setting Up the EPR Spectrometer and Acquiring DEER Data

  • Liquid N 2or liquid helium
  • DEER sample in EPR tube ( protocol 2)
  • 5‐L Dewar flask
  • Bruker EleXsys E580 operating at X‐band with the ER 4118X‐MD‐4/ER 4118X‐MD‐5 dielectric ring resonator
  • Tube adapter

Basic Protocol 4: Analysis of DEER Data

  • MatLab software
  • DeerAnalysis2013 software
  • DeerSim software (in‐house software of the research group; available upon request)
  • Origin 8.5 software
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Literature Cited

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