Electron Paramagnetic Resonance (EPR) and Spin‐labelling

Abstract

In EPR the magnetic dipole of an electron interacts with an external magnetic field and can be excited by an oscillating magnetic field. Spin labelling involves use of extrinsic probes, nitroxides, attached to the molecule under study. The spectra of nitroxide probes are used to infer orientation, dynamics and distances between labelled sites.

Keywords: magnetic resonance; protein structure; protein dynamics

Figure 1.

Spin labels used in the covalent modification of proteins: (a) maleimide; (b) methyl thiosulfonate; (c) ATP spin label.

Figure 2.

Energy level diagram of the Zeeman and hyperfine interactions for a nitroxide (Sz = ±½, Iz = 0, ±1). The vertical arrows denote EPR transitions with the resulting first‐derivative spectrum below. The g‐factor defines the centre of the resonance split by A due to hyperfine interactions with the nucleus.

Figure 3.

Orientational sensitivity of EPR spectra. (a) Splitting of the spectrum changes when the z‐axis of the nitroxide rotates with respect to the magnetic field. The centre of the spectrum changes when the nitroxide rotates about any axis. (b) Broadening of the spectra with increasing disorder: top, Gaussian disorder of ±1°; centre, ±5°; bottom, isotropic distribution.

Figure 4.

Orientation of the catalytic domain of the myosin head in the intermediate stages of the contractile cycle. Left to right: The myosin heads are dynamically disordered when detached from actin; initial binding in the presence of ATP provides little orientational and dynamic restraint; prepower stroke, actomyosin⋅ADP⋅Pi are still disordered but less mobile, postpower stroke, heads are rigid and stereospecifically bound to actin.

Figure 5.

(a) Sensitivity of the conventional EPR spectra to rotational motion and (b) ST‐EPR spectra. Note how the sharp resonances of the motionally averaged spectra progressively broaden, with the effect greatest in the high‐field region. In ST‐EPR spectra, decreasing motion increases the intensity at L″, C′ and H″ positions.

Figure 6.

Patterns of solvent accessibility for (a) β strands (2‐residue periodicity) and (b) an α helix (3.6‐residue periodicity).

close

References

Adhikari BB and Fajer PG (1996) Myosin head orientation and mobility during isometric contraction: effects of osmotic compression. Biophysical Journal 70: 1872–1880.

Altenbach C, Flitsch SL, Khorana HG and Hubbell WL (1989) Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines. Biochemistry 28: 7806–7812.

Perozo E, Cortes DM and Cuello LG (1999) Structural rearrangements underlying K+‐channel activation gating. Science 285: 73–78.

Raucher D, Sar CP, Hideg K and Fajer PG (1994) Myosin catalytic domain flexibility in MgADP. Biochemistry 33: 14317–14323.

Stone TJ, Buckman T, Nordio PL and McConnell HM (1965) Spin‐labeled biomolecules. Proceedings of the National Academy of Sciences of the USA 54: 1010–1017.

Thomas DD and Cooke R (1980) Orientation of spin‐labeled myosin heads in glycerinated muscle fibers. Biophysical Journal 32: 891–906.

Thomas DD, Ishiwata S, Seidel JC and Gergely J (1980) Submillisecond rotational dynamics of spin‐labeled myosin heads in myofibrils. Biophysical Journal 32: 873–889.

Further Reading

Berliner L and Reuben J (1989) Spin Labeling Theory and Applications, vol. 8. New York: Plenum Press.

Berliner L (1998) Spin Labeling: The Next Millennium, vol. 14. New York: Plenum Press.

Hubbell WL, Gross A, Langen R and Lietzow MA (1998) Recent advances in site‐directed spin labeling of proteins. Current Opinion in Structural Biology 8: 649–656.

Hustedt EJ and Beth AH (1999) Nitroxide spin–spin interactions: applications to protein structure and dynamics. Annual Review of Biophysics and Biomolecular Structure 28: 129–153.

Lichtenstein GI (1993) Biophysical Labeling Methods in Molecular Biology. Cambridge: Cambridge University Press.

Millhauser GL, Fiori WR and Miick SM (1995) Electron spin labels. Methods in Enzymology 246: 589–610.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Fajer, Peter G(Feb 2002) Electron Paramagnetic Resonance (EPR) and Spin‐labelling. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002985]