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
- 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:
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:
W.L., D.S. Cafiso, and C.A. Altenbach, Identifying
conformational changes with site-directed spin labeling. Nat.
Struct. Biol., 2000. 7:
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.
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:
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.
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
data. Biophys. J., 1997. 74: p. 1861-1877.
C., K. Cai, J. Klein-Seetharaman, H.G. Khorana, and W.L.
of inter-residue distances in spin labeled proteins at physiological
strategies and practical limitations. Biochemistry,
2001. 40: p. 15471-15482.
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.
G., Distance measurements
in the nanometer range by pulse EPR. ChemPhysChem, 2002. 3(11): p. 927-32.
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.