Membrane proteins and spin labelling

Mobility analysis of nitroxides in spin labeled protein.
EPR in conjunction with site-directed spin labeling (SDSL) is a technique to study structure and dynamics of membrane proteins. EPR provides information about the local environment of the spin label that has unpaired electrons but also can measure inter-spin label distances when two spin labels are introduced inside the protein.

Crystal structure of P-glycoprotein (3G5U.pdb)
Crystal structure of P-glycoprotein (3G5U.pdb)
EPR spectra of each attached nitroxide molecule showing correlation between position and mobility. Data courtesy of Prof. T. Prisner, University of Frankfurt (JACS, 2012, 134, pp 5857-5862)


EPR characterization of paramagnetic centers in cytochrome c oxidase.
Approximately 30% of all known proteins are metalloproteins. They are involved in a variety of biologically important processes such as electron transfer, drug metabolism, disease mechanisms, etc. EPR has an important role not only to study the electronic structure of metalloproteins but also to characterize their redox cofactors, binding sites, substrate reactions. For example, cytochrome c oxidase is a terminal protein in the respiratory chains from mitochondria and many bacteria. A low-spin heme, heme a, accepts electrons from a copper A (CuA) center bound to subunit II, and transfers them to a binuclear center.

Crystal structure (1OCC.pdb) of cytochrome c oxidase
EPR spectrum of cyt c oxidase showing heme a, copper A, and protein radical (in blue). Data courtesy of Prof. T. Prisner, University of Frankfurt (J. Phys. Chem. B, 2007, 111 (14), pp 3839–3846)

Enzyme Reactions

Detection and study of the active site of Cu,Zn-SOD.
Many enzyme reactions involve one-electron oxidation steps with formation of paramagnetic transient state of the enzyme detectable by EPR. The paramagnetic center where the unpaired electron is located, is usually centered at a transition metal (metalloproteins) or is an amino acid derived radical. Detection and identification of the paramagnetic centers is important to understand the function of the enzymes. For example, in the native SOD1 enzyme, the active site contains one Cu(II) ion that gives a very characteristic EPR spectrum.

RNA and DNA oxidation

DNA-derived radicals detected upon CuCl2/H2O2 treatment.
EPR spectroscopy in conjunction with spin trapping has been employed successfully to detect and identify high-molecular-weight species generated as a result of reactive oxygen species (ROS)-induced damage to biological macromolecules, such as DNAs and RNAs. The destruction or alteration of these materials is known to play a key role in a large number of cellular injuries and diseases.

EPR spectrum of N-centered radical upon DNA damage after CuCl2/H2O2 treatment using DMPO as spin trap. Spectrum also consists of two other radicals that are not DNA-derived. Data courtesy of Dr. R. Mason, NIEHS (Free Radic. Biol. Med. 2011 50(11) pp 1536
Mechanism of DNA damage by reactive oxygen species (ROS)

Screening DNP agents

EPR spectrum and dipolar coupling determination of bis-TEMPO.
Correct concentration of DNP polarizing agents is crucial to the success of a DNP experiment. Samples can be pre-screened before DNP experiments using the patented SpinCount module, even in the MAS rotor. Relaxation times are critical for DNP efficiency therefore P1/2 measurements at low temperature to estimate the DNP efficiencies of new polarization agents are invaluable. Another characteristic of importance in DNP measurements is the electron-electron dipolar coupling that is easily measured from solution and frozen solution EPR spectra. Read more about DNP agents

EPR spectrum of DNP agent (bis-TEMPO biradical)

D [G] = 18562/R3 [Å]

Dipolar coupling measured: D = 7.9 G. Distance determined: R = 13 Å

Data courtesy of Prof. T. Prisner, University of Frankfurt (Angew. Chem. Int. Ed., 2009, 48, 4996)

Crystal structure (1E9P.pdb) of Cu(II)-SOD protein
EPR spectrum of Cu(II)-SOD protein at 77 K using finger dewar
Contact an Expert to request for demo