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

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Site‐Directed Spin Labeling of Soluble Proteins

Materials

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

Materials

Soluble protein (e.g., HIV‐1 protease)
Deuterated sodium acetate (D₃ ‐NaOAc, 99%)
Deuterium oxide (D₂ O, 99%)
Deuterated glycerol (D₈ ‐glycerol, 99%)
Liquid N₂

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 N₂

Additional reagents and equipment for concentration or protein samples (unit 4.4 )

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

Materials

Liquid N₂ or 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

Materials

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

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Figures

Figure 17.17.1 Chemical structures of four common nitroxide spin labels before and after reacting with a cysteine side chain. (A ,B ) MTSL: (1‐oxyl‐2,2,5,5‐tetramethyl‐Δ3‐pyrroline‐3‐methyl) methanethiosulfonate; (C ,D ) IAP: 3‐(2‐iodoacetamido)‐proxyl; (E ,F ) MSL: 4‐maleimido‐TEMPO; and (G and H) IASL: 4‐(2‐iodoacetamido)‐TEMPO. The rectangular box represents the protein backbone.

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Figure 17.17.2 (A ) Four‐pulse DEER. Each pulse delay labeled with τ remains constant while spacing labeled with T is incremented. (B ) Pump and observe frequencies for nitroxide labels at X‐band.

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Figure 17.17.3 Sample dipolar evolution curve before (gray solid line) and after (black solid line) applying the background subtraction function (blue dashed line). The red line is the regenerated echo curve from data analysis, which is discussed in .

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Figure 17.17.4 Plots of the maximum spin concentration as a function of the inter‐spin distance. Solid line corresponds to the restriction imposed by instantaneous diffusion, while dashed line corresponds to the constraint imposed by intermolecular distances.

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Figure 17.17.5 Sample preliminary experiments prior to DEER experiment setup. (A ) Field‐swept spectrum to determine center field; (B ) echo decay experiment to measure phase‐memory time, T _m ; (C ) DEER echo acquisition to determine d0 and gate parameters; (D ) field‐swept echo‐detected spectrum to determine pump and observe frequencies. These data were collected at 65 K.

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Figure 17.17.6 The four‐pulse DEER sequence with the pulse spacings labeled according to Bruker Xepr software package nomenclature. In reference to the four‐pulse DEER sequence in Figure , d1 = τ₁ and d2 = τ₂ (or τ_max ). The time increment parameter dx is calculated as d2/ N , where N is the number of real data points. The start position of the echo signal is assigned as d0, while d3 is a critical delay parameter that prevents overlapping of the second observe pulse and pump pulse.

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Figure 17.17.7 Schematic diagram of three methods utilized for obtaining distance information from the background‐subtracted dipolar evolution curve.

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Figure 17.17.8 Example of an L‐curve (A ) and the corresponding distance profiles and dipolar modulation curves for low (B ,C ), optimal (D,E ), and high (F ,G ) regularization parameters (α).

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Figure 17.17.9 Self‐consistent procedure for determining the appropriate level of background subtraction.

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Figure 17.17.10 Effect of the breadth and most probable distance on the dipolar modulation curves. (A ) Varying FWHM of distance profiles centered at 36 Å; and (B ) the corresponding theoretical dipolar evolution curves with different decay rates. (C ) Profiles with different center distances but same FWHM and (D ) corresponding theoretical dipolar evolution curves with different oscillation frequencies. Inset highlights dipolar modulations that decay within the first 2 µsec.

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Figure 17.17.11 The influence of τ_max length on the corresponding distance profile. (A ) Gaussian distance profiles with most probable distance of 48 Å with FWHM of 1 Å (solid) and 7 Å (dashed). (B ) The corresponding theoretical dipolar evolution curves. Solid vertical line corresponds to τ_max of 2 µsec (C ) The same dipolar evolution curves as in (B), but τ_max is shortened to 2 µsec. (D ) The corresponding TKR distance profiles for (C), analyzed using DeerAnalysis software.

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Figure 17.17.12 DEER data for Subtype B HIV‐1 protease acquired at variable τ_max . (A ) Background‐subtracted dipolar evolution curves and the corresponding TKR fits (gray solid line). (B ) The corresponding TKR distance profiles. The vertical dashed line marks the position for a minor population centered at ∼40 Å. Data are vertically offset for clarity.

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Figure 17.17.13 Intensity‐normalized T _m curves and corresponding exponential decay fits for MTSL‐labeled HIV‐1 protease in 2 mM sodium acetate, pH 5.0, with H₂ O and 30% glycerol (dotted line), 2 mM sodium acetate buffer pH 5.0 with H₂ O and 30% deuterated glycerol (dashed line), 2 mM sodium acetate buffer pH 5.0 with D₂ O and 30% deuterated glycerol (solid line). The oscillations in the deuterated solvents originate from ESEEM effects between the deuterons and spin labels. The vertical dashed line marks τ = 3 µsec.

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Figure 17.17.14 DEER data processing for an HIV‐1 protease sample. (A ) Determination of zero time (x_c ) by fitting a Gaussian function to the –300‐ to +300‐nsec region of the echo curve. (B ) Raw dipolar echo curve and the exponential decay function (red solid line) corresponding to a homogeneous three‐dimensional distribution that is employed for background subtraction. (C ) Long‐pass filtered and background‐subtracted dipolar modulation curve with Tikhonov regularization (TKR) fit (red solid line) overlaid with Gaussian‐reconstructed dipolar modulation (blue solid line). (D ) The L‐curves derived from TKR analysis that helps determine the optimal regularization parameter (α). (E ) TKR distance profile overlaid with the linear combination of Gaussian populations (red dashed line). Peaks labeled with asterisk indicate populations ≤5% that are not statistically significant at 95% confidence level based on the χ² criterion. (F ) The Gaussian populations employed to regenerate the TKR distance profile. (G ) The Pake dipolar pattern that results from the Fourier transformation of the background‐subtracted dipolar modulation curve and the corresponding fit (red dashed line). (H ) Table of values for the most probable (center) distances, full width at half maxima (FWHM), and relative percentage of conformational populations (Pop. %) used for generating the Gaussian‐reconstructed distance profile in E (red dashed line).

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