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.

Solid phase synthesis and spectroscopic characterization of the active and inactive forms of bacteriophage S21 pinholin protein

“…CW-EPR spectroscopy of these spin-labeled molecules can reveal information about the motion of the nitroxide side chain, solvent accessibility, and the polarity of the surrounding environment. [21,33] The successful spin labeling of the pinholin protein and incorporation into both bicelle and MLV lipid mimetic systems is shown in the CW-EPR spectra in Figure 5. This EPR data was also used to calculate spin labeling efficiency for each peptide which ranged from ~85-90% labeling efficiency.…”

“…This decrease in the motion of the MTSL will broaden the lines in the EPR spectra and cause a decrease in their amplitude which is present in Figure 5B. [33,34] The broadening of the EPR spectral linewidth is quantitatively determined by measuring the central line width. In Figure 5A the free MTSL spectra shows a central line width of 1.4 G, while the MTSL bound to the pinholin, 5B, has a central line width of 2.9 G. The line broadening of the EPR spectra from Figure 5A to 5B confirms the successful disulfide bond formation between the free MTSL and the Cys side chain of the pinholin due to a more restricted environment for the bound SL.…”

Protein Conformational Dynamics upon Association with the Surfaces of Lipid Membranes and Engineered Nanoparticles: Insights from Electron Paramagnetic Resonance Spectroscopy

“…In nitroxide-labeled proteins lacking native paramagnetic centers, CW EPR records only the absorption spectrum of the nitroxide radical, usually in the form of the first derivative. The spectrum exhibits characteristic three lines, whose linewidths and amplitudes depend on the intrinsic spin label dynamics caused mainly by the rotational diffusion of the nitroxide moiety in the local potential and to some extent by the protein dynamics in its immediate vicinity as well as the protein global tumbling, segmental motion or chemical exchange [ 52 , 53 , 54 ]. In general, the frequencies of these motions observed by nitroxide multifrequency CW EPR fall into the range of rotational correlation time (τ c ) ≅ 10 −12 –10 −8 s. The anisotropy of hyperfine interaction (hfi) tensor and g-tensor, partly averaged by motions, produces typical asymmetric spectrum with the high-field side being broader.…”

Simulation Best Practices for Lipid Membranes [Article v1.0]

“…First, diffusivities can be determined from a variety of methods, including fluorescence techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS), quasi-elastic neutron scattering, EPR, and NMR, amongst others. The length and time scale of study can vary significantly with different methods; for example, EPR and NMR (O(1 nm-100 µm),O(1 ns-1 ms)) [150][151][152][153] and quasi-elastic neutron scattering (O(0.1-10 nm,<1 ns)) [44]. Furthermore, neutron spin echo spectroscopy has recently been shown to probe ∼100 ns and obtain membrane viscosity estimates that can be used to estimate in-plane diffusivity [154].…”