Site-Directed Spin Labeling EPR for Studying Membrane Proteins

Abstract

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a rapidly expanding powerful biophysical technique to study the structural and dynamic properties of membrane proteins in a native environment. Membrane proteins are responsible for performing important functions in a wide variety of complicated biological systems that are responsible for the survival of living organisms. In this review, a brief introduction of the most popular SDSL EPR techniques and illustrations of recent applications for studying pertinent structural and dynamic properties on membrane proteins will be discussed.

Figure 1

Structure of nitroxide spin labels used in the SDSL EPR study of micromolecules. (a) Methanethiosulfonate spin label (MTSL), (b) maleimide spin label (MSL) N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide, (c) iodoacetamide spin label (ISL), (d) bis(1-oxyl-2,2,5,5-tetramethyl-3-imidazolin-4-yl) disulfide (IDSL), (e) bifunctional spin label (BSL), (f) 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid (TOAC), and (g) 4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP).

Figure 2

Structure of MTSL (methanethiosulfonate spin label) and the resulting side-chain produced by reaction with a cysteine residue on a protein.

Figure 3

Energy level diagram of a nitroxide spin label in the presence of a magnetic field B₀. The lower panel shows the corresponding EPR spectra in the absence and presence of a ¹⁴N (I = 1) hyperfine interaction.

Figure 4

(a) The proposed topology of the KCNE1 sequence in lipid bilayers. (b) Plot of inverse central EPR resonance linewidth (m_I = 0) as a function of residue position of KCNE1 in lipid bilayers (adapted from [51] with permission).

Figure 5

Three-pulse ESEEM FT data of ²H-labeled d₁₀ Leu4 LRL8 peptide in TFE (α-helix) and DPPC liposomes (3₁₀-helix) for i + 3 and i + 4 samples (adapted from [52] with permission).

Figure 6

CW dipolar broadening EPR study of KCNE1. (a) CW dipolar broadening EPR spectra of KCNE1 bearing BSLs at sites 53/57 and 63/67 in POPC/POPG liposomes (left panel) and the corresponding distance distribution (right panel) obtained from data analysis by using the Short Distances LabVIEW program. (b) Cartoon representation of KCNE1 bearing two BSLs at sites 53/57 and 63/67 (left panel) and the corresponding distance distribution obtained from 20 ns molecular dynamics trajectory data analysis (right panel) (adapted from [53] with permission).