EPR - Bruker E500

Bruker E500 spectrometer used for X-band (≈9.5 GHz) CW EPR spectroscopy.

The vast majority of biological macromolecules are diamagnetic (i.e., no molecular orbital is singly occupied by an electron). Electron paramagnetic resonance (EPR) spectroscopy is a technique applied to paramagnetic species (i.e., chemicals with unpaired electrons) in order to characterize their molecular frameworks and the chemical environments around them through the eyes of unpaired electrons.

Proteins can also be investigated in this manner via site-directed spin labeling (SDSL). This is when paramagnetic (or “spin”) centres are covalently linked to proteins at specific amino acid residues.

Cysteine sub image

Cysteine modification used in site-directed spin labeling.

SDSL EPR experiments can be used to uncover substantial amounts of site-specific biophysical information about a protein (e.g., relative mobilities of, the solvent accessibilities of, and distances between amino acid side chains), which ultimately unearths details about the dynamics of said protein (i.e., what physically happens when it “works”).

Common site-directed spin labeling is based on cysteine substitution mutagenesis and reaction of the unique cysteine with a sulfhydryl-specific spin label to introduce a disulfide-linked nitroxide side chain.

EPR analysis example

4-hydroxy-TEMPO in 90% glycerol, measured at various temperatures.

On the right, a model nitroxide radical, 4-hydroxy-TEMPO in 90% glycerol, measured at various temperatures in order to illustrate the effect of the rotational correlation time on spectral anisotropy. When a nitroxide radical’s motion becomes more hindered (by steric interactions in the case of spin-labeled proteins), the result will be that the parameters of the CW EPR spectrum will shift towards those of the maximally anisotropic powder spectrum.

Equipment

1) Continuous wave (CW) EPR spectroscopy can measure the absorption and relaxation properties of spin labels on spin-labeled proteins, thereby yielding information about the mobility, solvent accessibility, polarity, etc. of the labeled site. Moreover, CW EPR can be used to measure short distances (<25 Å) between spin labels, and assay the quantity of unbound (“free”) label in solution, which is considered a contaminant in pulse EPR experiments. Typically, 5–8 µL of a ~25 µM protein sample is sufficient for these measurements.

2) Double electron–electron resonance (DEER) spectroscopy is a pulse EPR experiment that measures long distances (15 Å–80 Å) between spin labels. DEER has gained much popularity due to its ability to yield individual distance in the population. Typically, 15 µL of a 50–100 µM protein sample is sufficient for these measurements at 80 K. Samples are often supplemented with glycerol as a cryoprotectant.

Bruker E580 spectrometer used for Q-band (≈34 GHz) DEER EPR spectroscopy

Bruker E580 spectrometer used for Q-band (≈34 GHz) DEER EPR spectroscopy.

Location

Medical Sciences Building, Rooms 5252 and 1204

Coordinator and Contact

Oliver P. Ernst

Oliver P. Ernst

MSB, Room 5259
1 King's College Circle
416-978-3849
oliver.ernst@utoronto.ca