Site-directed spin labeling.

Our work on transport, membrane fusion and cell-signaling requires both static and dynamic information on membrane proteins. A particularly promising approach to obtain detailed information on these systems is an emerging electron paramagnetic resonance (EPR) based technique termed site-directed spin labeling (SDSL).This method combines site-directed mutagenesis with chemical labeling to replace a native protein side chain with a nitroxide side chain.The most widely utilized labeling scheme is shown below, where site-specific cysteines are derivatized with a methanethiosulfonate (MTSL) label to produce the side chain R1.

Remarkably, the EPR spectra of the nitroxide R1 are uniquely defined by protein secondary structure, backbone motion, and tertiary contact (1-3).A direct measurement of the frequency of collisions between a secondary paramagnetic species (such as oxygen) and R1 may be made through saturation techniques, providing a direct measure of solvent accessibility and membrane depth of the R1 side chain (4).In double labeled proteins, dipolar interactions between pairs of labels yields long-range distances (6 to 25 Angstroms) (5-8). These distance measurements may be extended dramatically, out to 50 Angstroms or more, with newer pulse methods such as double electron-electron resonance (DEER) (9,10).



SDSL is an exciting new methodology that has been successfully applied to a number of important protein systems. It is relatively inexpensive, requires modest amounts of protein, has no molecular weight limitations and can be carried out in a wide range of environments (from intact cells to reconstituted membranes or micelles). The incorporation of R1 is also well-tolerated in proteins (11).

References on site-directed spin labeling.

  1. Hubbell, W.L. and C. Altenbach, Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr. Op. Struct. Biol., 1994. 4: p. 566-573.
  2. Hubbell, W.L., A. Gross, R. Langen, and M.A. Lietzow, Recent advances in site-directed spin labeling of proteins. Curr. Op. Struct. Biol., 1998. 8: p. 649-656.
  3. Hubbell, W.L., D.S. Cafiso, and C.A. Altenbach, Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol., 2000. 7: p. 735-739.
  4. Altenbach, C., D.A. Greenhalgh, H.G. Khorana, and W.L. Hubbell, A collision gradient-method to determine the immersion depth of nitroxides in lipid bilayers.Application to spin-labeled mutants of bacteriorhodopsin. Proceedings of the National Academy of Sciences, USA, 1994. 91: p. 1667-1671.
  5. Rabenstein, M.D. and Y.-K. Shin, Determination of the distance between two spin labels attached to a macromolecule. Proc. Natl. Acad. Sci. USA, 1995. 92: p. 8239-8243.
  6. Steinhoff, H.-J., N. Radzwill, W. Thevis, V. Lenz, D. Brandenburg, A. Antson, G. Dodson, and A. Wollmer, Determination of interspin distances between spin labels attached to insulin:comparision of electron paramagnetic resonance data with the x-ray structure. Biophys. J., 1997. 73: p. 3287-3298.
  7. Hustedt, E.J., A.I. Smirnov, C.F. Laub, C.E. Cobb, and A.H. Beth, Molecular distances from dipolar coupled spin-labels:the global analysis of multifrequency continuous wave electron paramagnetic resonance data. Biophys. J., 1997. 74: p. 1861-1877.
  8. Altenbach, C., K. Cai, J. Klein-Seetharaman, H.G. Khorana, and W.L. Hubbell, Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations. Biochemistry, 2001. 40: p. 15471-15482.
  9. Borbat, P.P., H.S. McHaourab, and J.H. Freed, Protein structure determination using long-distance constraints from double-quantum coherence ESR: study of T4 lysozyme. J. Am. Chem. Soc., 2002. 124(19): p. 5304-14.
  10. Jeschke, G., Distance measurements in the nanometer range by pulse EPR. ChemPhysChem, 2002. 3(11): p. 927-32.
  11. Mchaourab, H., M. Lietzow, K. Hideg, and W. Hubbell, Motion of spin-labeled side-chains in T4 lysozyme. (I) Correlation with protein structure and dyanmics. Biochemistry, 1996. 35: p. 7692.