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High-field CW EPR with Gd(III) spin labels for structure studies of membrane proteins

  • Author(s): Clayton, Jessica Ann
  • Advisor(s): Sherwin, Mark S
  • et al.
Abstract

Electron paramagnetic resonance (EPR) in combination with site-directed spin labeling (SDSL) is a powerful tool for elucidating the structure, organization, and dynamics of biomolecules in native-like environments. With EPR and SDSL, we can site-specically label pairs of sites in a biomolecule and accurately measure the distance, or distribution of distances, between them on length scales ranging from Angstroms to several nanometers. Of particular interest are membrane proteins and higher-order membrane protein complexes, which have historically resisted traditional biophysical characterization techniques. EPR as a means to measure protein structure becomes even more powerful at high fields and using Gd(III) spin labels, which together provide much improved sensitivity. This work expands on the capabilities of high-field continuous-wave (CW) EPR for distance measurement with spin labels based on Gd(III) complexes. First, we investigate a model system of and show that CW EPR with Gd(III) labels allows for distance measurements in the range of at least 1.2 - 3.4 nm at cryogenic temperatures. We additionally show that distance measurements are possible up to room temperature. Next, we investigate the zero-field splitting - a property of great importance for determining the EPR lineshape of high-spin systems - for a variety of different Gd(III) complexes. Combining EPR spectra measured at 35 GHz, 95 GHz, and 240 GHz, we compare literature models for the broadly distributed second-order ZFS parameters D and E. We test these results against a superposition model for predicting the magnitude of the ZFS based on knowledge of the structure of a Gd(III) complex, which can potentially be useful for designing new Gd(III) complexes tailored for use as spin labels with high-field EPR. Finally, we apply high-field CW EPR with Gd(III) spin labels to the study of proteorhodopsin (PR), a transmembrane protein that functions as a light-driven proton pump for marine bacteria. Inter-PR CW EPR distance measurements in the range of ~1.5 - 3 nm are used to elucidate the functionally relevant oligomeric structure of PR, demonstrating the usefulness of this technique in targeting complex oligomeric systems. Finally, we present the development of methods which will allow CW EPR with Gd(III) to be used as a probe of protein dynamics, by measuring at a distance change induced by motions of the E-F loop region of PR upon light activation.

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