Protein Structural Dynamics Revealed by Site-Directed Spin Labeling and Multifrequency EPR

Abstract

Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

“…However, with the development of site-directed spin-labeling (SDSL) techniques to target biological systems, there has been a significant increase in the application of EPR spectroscopy to study protein structure and dynamics (Alexander, Bortolus, Al-Mestarihi, Mchaourab, & Meiler, 2008; Altenbach, Flitsch, Khorana, & Hubbell, 1989; Fanucci & Cafiso, 2006; Hirst, Alexander, McHaourab, & Meiler, 2011; Hubbell, Gross, Langen, & Lietzow, 1998; Hubbell, López, Altenbach, & Yang, 2013; Sahu, McCarrick, & Lorigan, 2013; Sahu, McCarrick, Troxel, et al, 2013). SDSL EPR is sensitive to dynamics on the picoseconds to microsecond timescales, which cover a wide range of motions in biological and molecular systems (Barnes, Liang, Mchaourab, Freed, & Hubbell, 1999; Casey et al, 2014; Nesmelov, 2014). Also, the topology of a membrane protein can be explored with respect to the lipid bilayer with SDSL coupled with CW-EPR spectroscopy.…”

Investigation of Liquid‐Phase Inhomogeneity on the Nanometer Scale Using Spin‐Polarized Paramagnetic Probes

“…The dominant feature here is the m N ¼AE ½ doublet from the 13 C-benzoyl radical, with a hyperfine coupling constant on the 13 Ccarbonyl,A( 13 C) ¼ 127 G. Each line in this doublet is a superposition of the APS from the SCRP and ST 0 RPM polarized signal from free (escaped) radicals. In the latter, the m N ¼þ1/ 2l i n eisi nem i s si on ;th ecor re s po nd i n gm N ¼À½ line is in absorption (the E/A pattern).…”

“…Here, we present an example that demonstrates the high sensitivity observed for the APS in the 13 C-benzoyl radical as a function of the size of its alkyl sulfate micellar host. Moreover, it has been found that a decrease in micelle size results in a strong asymmetry of the observed APS.…”

“…An example of the APS feature in a TREPR spectrum is given in Figure 4, where spectrum A belongs to a radical pair (Scheme 2) generated by the laser flash photolysis of 13 C-labeled in the carbonyl group 2,2-dimethyl-desoxy-benzoin (DMDB) in an aqueous micellar solution of SDS at room temperature. Figure 4 belongs to the same radical pair acquired at the same t obs ¼ 360 ns and under the same experimental conditions (temperature, microwave power, etc.)…”

“…Modern development of the method is deeply connected to recent advancements in EPR instrumentation. Examples include an increase in the microwave frequency [8,9] up to 215-240 GHz (very high frequency EPR, VHF/EPR), application of pulsed EPR spectrometers [9,10], as well as advances in targeted spin labeling (site-directed spin labeling, SDSL [11][12][13]). New chemical synthesis methods have resulted in novel probe structures designed, for example, to measure the polarity of the microenvironment surrounding the probe [14], the concentration of oxygen [15], and other properties in both homogeneous and heterogeneous systems.…”