Development of Gadolinium(III) spin labels to study structure and dynamics of membrane proteins

Figure 1: Spin labeling.  SDSL is accomplished by introducing an EPR-active side chain at selected locations in proteins. For example, by attaching a Gd3+ based spin label on residues of interest in proteorhodhopsin.
Membrane proteins control the flow of matter, energy, and information between the interior and exterior of a cell and are of fundamental importance to a wide range of cellular processes. Despite their prevalence, membrane proteins remain poorly understood due to their notorious resistance to standard NMR and X-ray crystallography techniques. An emerging technique, electron paramagnetic resonance (EPR) in conjunction with site-directed spin labeling (SDSL) facilitates elucidation of protein structure by allowing measurement of distances between selectively labeled sites on a protein or protein complex. This technique has several advantages over conventional NMR, including sensitivity of absolute number of spins, spatial resolution for spin-spin distance measurements, and time resolution of dynamical structure change.

The goal of this project is to develop a new class of spin labels based on the spin-7/2 Gd3+ ion to enable structural studies under a variety of biologically important conditions that are difficult or impossible with conventional nitroxide spin labels. The paramagnetic core of the Gd3+ ion is well shielded by outer electrons, making it much less sensitive to the local chemical environment and will survive for days in vivo. Unlike nitroxides, Gd3+ spectra are relatively insensitive to nearby protons, allowing for EPR in environments that are not deuterated. Distance measurements can be performed on samples less than 1/10th the volume of samples using nitroxides, enabling study of proteins that are difficult to express in large quantities. Additionally, with continuous wave EPR using Gd3+ based spin labels we are able to achieve superior sensitivity for making distance measurements at biologically relevant temperatures. The paramagnetic attributes of the Gd3+ ion are particularly favorable at high magnetic fields and frequencies, one of the frontiers of EPR.

Figure 2: PR hexamer.  Model of proteorhodopsin hexamer, with distances measured using a Gd3+-based spin label and high-field EPR
Currently, we are particularly interested in studying structure and conformational changes in proteorhodopsin (PR), a recently discovered light-activated proton pump found in marine bacterioplankton. PR belongs to the large class of the heptahelical trans-membrane (7TM) proteins, which make up 40% of all drug targets.

UCSB Researchers:
Mark Sherwin (Physics)
Prof. Songi Han (Chemistry and Biochemistry)
Graduate Students:
Blake Wilson (Physics)
  1. Edwards, Devin T.; Ma, Zhidong; Meade, Thomas J.; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S. Extending the distance range accessed with continuous wave EPR with Gd3+ spin probes at high magnetic fields, Physical Chemistry Chemical Physics, 15, 11313-11326 (2013) [www]
  2. Edwards, Devin T.; Huber, Thomas; Hussain, Sunyia; Stone, Katherine M.; Kinnebrew, Maia; Kaminker, Ilia; Matalon, Erez; Sherwin, Mark S.; Goldfarb, Daniella; Han, Songi Determining The Oligomeric Structure Of Proteorhodopsin By Gd3+-Based Pulsed Dipolar Spectroscopy Of Multiple Distances Structure, 22, 1677 (2014) [www]
  3. Clayton, Jessica A.; Qi, Mian; Godt, Adelheid; Goldfarb, Daniella; Han, Songi; Sherwin, Mark S. Gd3+-Gd3+ Distances Exceeding 3 Nm Determined By Very High Frequency Continuous Wave Electron Paramagnetic Resonance Physical Chemistry Chemical Physics, 19, 5127 (2017) [www]