On the relationship between distance information derived from cross-linking and from resonance energy transfer, with specific reference to sites located on myosin heads.
The techniques of fluorescence resonance energy transfer (FRET) and cross-linking can provide complementary information concerning the relative separation of a pair of sites. Cross-linking experiments provide an assessment of the distance of closest approach between a pair of sites. FRET measurements, by contrast, yield information about the average distance between the pair of sites. We have taken advantage of hybrid myosins to understand the relationship between distances obtained for a pair of equivalent sites, one on each myosin head, using both FRET (steady-state and time-decay) and cross-linking techniques. The rigid cross-linker, 4-4'-dimaleimidyl-stilbene-2-2'-disulfonic acid (DMSDS), can efficiently cross-link the two myosin regulatory light-chains, each at residue Cys50 of the Mercenaria regulatory light chain (Chantler, P.D., and S. M. Bower. 1988. J. Biol. Chem. 263:938-944), indicating that these sites can come within 18 +/- 2 A of each other. In a complementary set of experiments, steady-state and time-decay measurements using fluorescence donor/acceptor pairs located at these same sites indicate transfer efficiencies of somewhat less than 20%, suggesting an average separation of greater than 50 A between sites (Chantler, P. D., and T. Tao. 1986. J. Mol. Biol. 192:87-99). Here, we present theoretical calculations which show that efficient cross-linking can be achieved readily in dynamic systems such as the heads of myosin, even though the necessary subpopulation of proximate molecules at any instant may be below the detection limits of time-decay-FRET. Therefore, cross-linking experiments can provide important ancillary information about the extent of motions within a marcomolecular system when used in conjunction with FRET.As a corollary, demonstration of extensive cross-linking does not necessarily indicate a static proximity; the mean separation distance should be ascertained by other methods such as FRET.
Chantler
P D
PD
Department of Anatomy and Neuroscience, Medical College of Pennsylvania, Philadelphia 19129.
Tao
T
T
Stafford
W F
WF
D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D003432:Cross-Linking Reagents; D004735:Energy Transfer; D005453:Fluorescence; D066298:In Vitro Techniques; D008954:Models, Biological; D009218:Myosins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D009218:Myosins